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Polymerization amplified SPR−DNA assay on noncovalently functionalized graphene
Author’s Accepted Manuscript Polymerization Amplified SPR−DNA Assay on Noncovalently Functionalized Graphene Pei-Xin Yuan, Sheng-Yuan Deng, Chuan-Guang Yao, Ying Wan, Serge Cosnier, Dan Shan www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(16)30659-5 http://dx.doi.org/10.1016/j.bios.2016.07.031 BIOS8912
To appear in: Biosensors and Bioelectronic Received date: 30 November 2015 Revised date: 30 June 2016 Accepted date: 7 July 2016 Cite this article as: Pei-Xin Yuan, Sheng-Yuan Deng, Chuan-Guang Yao, Ying Wan, Serge Cosnier and Dan Shan, Polymerization Amplified SPR−DNA Assay on Noncovalently Functionalized Graphene, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.07.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Polymerization Amplified SPR−DNA Assay on Noncovalently Functionalized Graphene Pei-Xin Yuana, Sheng-Yuan Denga,*, Chuan-Guang Yaoa, Ying Wan,b Serge Cosniera,c，Dan Shana,* a
Sino-French Laboratory of Biomaterials and Bioanalytical Chemistry, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China b Intelligent Microsystem Technology and Engineering Center, School of Mechanical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China c University of Grenoble, Alpes−CNRS, DCM UMR 5250, F−38000 Grenoble, France
[email protected] [email protected] *
Corresponding author. Tel./Fax: +86−(25)−84303107
Abstract A highly efficient surface plasmon resonance (SPR)-based DNA assay was developed, by employing noncovalently functionalized graphene nanosheets as a substrate, and enzymatic catalysis-induced polymerization as mass relay. The objective of this strategy was manifold: first of all, to sensitize the overall SPR output by in situ optimized electrogeneration of graphene thin-film, which was characterized by atomic force microscopic topography; secondly, to regulate the self-assembly and orientation of biotinylated capture probes on nickel-chelated nitrilotriacetic acid (NTA) scaffolds, that anchored onto graphene-supported pyrenyl derivatives; and lastly, to synergize the signal amplification via real-time conversion of the additive aniline into polyaniline precipitation by horseradish peroxidase-tagged reporters. With this setup, a precise and replicable DNA sensing platform for specific targets was achieved with a detection limit down to femtomolar, thus demonstrating a beneficial exploration and 1
exploitation of two-dimensional nanomaterials as unique SPR infrastructure. The possibility of such ″bottom-up″ architecture mounted with ″top-down″ weight reactor would be most likely extensible and adaptable to protein determinations.
Keywords: surface plasmon resonance; DNA assay; electrochemically reduced graphene nanosheets; pyrene-tethered nitrilotriacetic acid; biocatalytic polymerization
1. Introduction
Owing to its intrinsic capability of interpreting near-interface heterogeneity, surface plasmon resonance (SPR) as an established analytical technology, has predominated in the study of ″bio-mass″ interaction and migration (Fang, et al., 2015; Stern, et al., 2014; Wijaya, et al., 2011). Comprehensively, dual aspects could be well exemplified in the manipulation of sequence-dependent DNA hybridization, which, in its very essence, embodies an immovable code for versatile utilizations on SPR platform, or more pertinently, the DNA biosensing (Cibulskis, et al., 2013; DeKosky, et al., 2013; Pattanayak, et al., 2013). Theoretically, oscillatory frequency, dielectric constant and proximal thickness comprise the primary variables for SPR signal transduction (Guo, 2012); while in realistic practices, far-end modulation towards the sensitization of SPR response became a strategic preference in terms of either composited probes as nano-resonators (Ding, et al., 2014; Krishnan, et al., 2011), or molecular biology, typically the terminal deoxynucleotidyl transferase extension, rolling cycle amplification, and polymerase chain reaction (Li, et al., 2011; Yuan, et al., 2015). On the other side, none could deny the fact that the load and layout of underlying capture units exert a most neglected profound influence on the explicit efficiency of standard 2
protocols (Cosnier and Holzinger, 2011; Guo, et al., 2013), where the dilemma also hides that the valid density of immobilization turned out below consensus by the plausible Au−S bonding (Jimenez-Monroy, et al., 2013; Tang, et al., 2014) on conventional SPR chips (Cai, et al., 2015; Song, et al., 2014). To circumvent such predicament, tectonically by observing the bottom-up development, reconstituting fine structures of the surface was motivated in recent years (Liu, et al., 2015; Subramanian, et al., 2014; Zagorodko, et al., 2014), infusing vigor and vitality in the fundamentals of SPR-centered bioanalysis. For instance, the transplant of tetrahedral DNA origami individuals accomplished an extraordinarily non-blocking, mechanically rigid, yet epitaxially flexible substrate (Pei, et al., 2010), particularly qualified for biometric identification (Kumar, et al., 2014). By contrast, in a holistic and macroscopic view, it is universally acknowledged that on-site large-scale overlay, by electrospun fibration (Camposeo, et al., 2015), LangmuirBlodgett filming (Geldmeier, et al., 2014), or chemical vapor deposition (Liu, et al., 2010; Losurdo, et al., 2014), emerged to be a relatively and increasingly more accessible and accountable approach. Therein, an incarnation of SPR immunoassay has been dedicated by virtue of polymethyl-methacrylate-assisted transfer of graphene-like two-dimensional nanomaterials (Chen, et al., 2015; Han, et al., 2015; Hou, et al., 2015; Xiang, et al., 2015; Yan, et al., 2015), precisely due to their enormously lamellar flatness and periodically planar homogeneity (Das, et al., 2011; Subramanian, et al., 2013). On the merit of these potent properties, decoration of densely populated gold nanoparticles (Xue, et al., 2014; Yang, et al., 2014; Zhang, et al., 2014; Zhu, et al., 2013), and noncovalent self-assembly of highly ordered scaffolds (Eigler and Hirsch, 2014), as fruitful measures for post-functionalization, further achieved unprecedented SPR enhancement in DNA and protein detections 3
(Wang, et al., 2015; Zagorodko, et al., 2014), which shall be retrospectively ascribed to the matter of frameworks at bottom. Hierarchically, this non-routine perspective could be assumed compatible and even able to synergize cohesively with rational top-level designs, hence consummating an ultimate pattern of SPR-based methodologies. Inspired by the above heuristic review and progressive insight, a tentative research focusing on the SPR reinforcement at both ends for the determination of oligonucleotides was proposed (Scheme 1), which commenced with in situ electrophoretic precipitation of graphene oxide (GO) in aqueous solution to a membrane of graphene nanosheets (rGO) as capacious infrastructure. As a preliminary, cyclic voltammogram (CV)-tandem SPR (ESPR) proceeded to depict simultaneously this facile modification; whereas microscopic imaging illustrated the stepwise upward constructions. To accommodate DNA probes at its best, also to regulate their alignment, abundant nickel-chelated nitrilotriacetic acids (NTA) were tethered onto rGO-supported pyrene groups (NTPy). This scaffolding fabric, with reported association (kon = (1.1 ± 0.5) × 106 / Ms) and dissociation (koff = (7.1 ± 1.1) × 10−4 / s) constants comparable to the bindings of streptavidin/biotin (Pia and Martinez, 2015), circumscribed the on-chip orientation and footholds allowed for only biotinylated biomolecules (cDNA) via axial ligation of Ni2+−S (nickel perchlorate or Ni(ClO4)2−biotin), rendering rather vast solid-state expression of targets (tDNA) than that on traditional monolayer of Au−thiols. Moreover, for the sake of stoichiometric cascade, a sandwich format was implemented stereochemically to bridge the capture with the bio-reporter (rDNA): horseradish peroxidase-labeled streptavidin (HRP-SA), enabling a dynamic biocatalytic polymerization of aniline the reductant into polyaniline (PANi) (Jiang, et al., 2014; Wang, et al., 2013; Xu, et al., 2015). As a 4
conductive polymer (Prathap, et al., 2012; Yu, et al., 2014a), its long-chains sank and intertwined gradually, overweighing its carrier and accordingly disturbing specific conductance in the neighborhood. Therefore, an ultrasensitive DNA sensor came into being by coupling double SPR-multipliers in altitudinal series. In this way, such integration of reform about pristine matrices with joint mass effects by trigger would symbolize a navigable highway towards the uncharted horizon of SPR-related bioapplications.
[Scheme 1]
2. Experimental section
Please consult Supporting Information for elaborations on catalogued experimentation and instrumentation, concerning: 1. Materials; 2. Measurements and Apparatus; 3. Electrodeposition of Graphene Nanosheets; 4. NTA-Intermediated Immobilization of cDNA on rGO Surface; 5. DNA Hybridization for Enzyme-Linked Oligonucleotide Sorbent Assay; and 6. Biocatalytic Polymerization-Triggered Mass Effect of Tracers. As a reminder, the flow injection was adopted as a primary sampling method for most situations throughout the overall protocol, while the static method was implemented exceptionally for the hybridization of tDNA segment.
3. Results and discussion
3.1. Characterization and optimization of rGO thin-film As elaborated before, the SPR output counts substantially on the volume of probes.
5
In view of that, rGO was selected, as a capping agent on the whole, to veil the ready-made golden face. First and foremost, this originated from its comparatively immense surface-to-volume ratio than that of the bare one (Wang, et al., 2011). More than that, the basal plane of this browny shroud appeared to be fairly susceptible of ″stains″ at molecular level (Subramanian, et al., 2013). And remarkably, it was testified that the graphene-plated gold-leaf rectifies the power streams across dielectric sub-coatings within and redirects the field of polaritons (Bao and Lok, 2012; Bonaccorso, et al., 2010). These structure-activity relationships stimulated the exploitation of rGO in SPR, which was fulfilled facilely by CV (see Supporting Information: 3. Electrodeposition of Graphene Nanosheets). As shown in Figure 1A, blue curves, two reducing peaks rose with approximate onsets at −0.75 and 0.1 V, respectively, whose corresponding currents escalated with the incremental circulation (Figure 1A, curve a: 1st and b: 500th loop), suggesting an inherent structural evolution of GO. That is, the successive decarboxylation at negative ranges, during which the suspended GO platelets accumulated in the diffusion layer and adsorbed onto the electrode because of depolarization and hydrophobicity (Harrison, et al., 2015; Zhang, et al., 2015). This kinetics was reflected in a frequency (ω, i.e. ΔAngle) vs. potential diagram (Figure 1A, red curves), where conspicuous SPR angles fluctuated drastically, echoing against the voltammetric behaviors around that sole redox pair at a formal potential of 0.18 V. Coincidentally, both galvanic and ESPR responses, whether belonging to summits or baselines, augmented globally (Figure 1A, curves a and b). Especially, the inflation in charging currents alongside the loops indicated the ongoing thickening of deposited rGO flakes (Liu, et al., 2015; Subramanian, et al., 2011). Once investigated in details, ESPR plots in Figure 1A was then translated into a time-elapsed procession of Figure 1B, from which one could witness the significant 6
effect of incessantly iterative oxidation-reduction of GO on the SPR reflectivity; otherwise it would be overloaded in the long haul. In this case, the thickness of rGO must be optimized in light of the cycles in question, which was interrogated by comparing feedbacks among prefactoring dsDNA/NTPy/rGO (past various depositing intervals, dsDNA: cDNA+tDNA). As a result, the SPR responses for tDNA hybridization surged in company with the prolongation of electro-precipitation, and climbed to the climax at 500 repetitions (Figure 1C), manifesting the attachment of rGO did favor the habitats for cDNA up to its saturation. Hereafter, the signal collapsed probably out of a side-effect that the transmittance of rGO tier grew too low for the incident light to penetrate. Thus, the number 500 was adopted as an appropriate value for counting down. As a supplementary, rGO film by this means of batch-production was visualized in field-emission scanning electron microscopic (FESEM) image (Figure 1D), where rGO fragmentation overlapped into a wrinkled landscape by thermodynamic perturbation (Kundu, et al., 2012; Yu and Park, 2014b), however, still possessing velvety texture, a superior edge for more binding opportunities over pure gold.
[Figure 1]
3.2. Downside fabrication of cDNA/NTPy/rGO and its hybridizing capacity The settling of cDNA in the conceived scenario (see Supporting Information: 4. NTA-Intermediated Immobilization of cDNA on rGO Surface) was demonstrated in Figure 2A. Brand-new gold disk displayed a basic blank of −1630 mdeg (m°) in ultrapure water (Figure 2A, line a). The coverage of rGO raised the SPR angle up to −815 mdeg (Figure 2A, line b), a clear evidence of the rGO/Au outcome. Furthermore, 7
the baseline of rGO/Au was lifted up to a new plateau at −375 mdeg by the addition of NTPy (NTPy/rGO/Au)(Figure 2A, line c), implying that the pyrene species in NTPy had adhered to the hexagonal lattices of graphene via noncovalent π-π stacking (Eigler, et al., 2014). In such occasion, the entry of Ni2+ would naturally register itself a guest-host inclusion by peripheral carboxyls of NTA for an upright direction of follow-up occupants (Baur, et al., 2010): the cDNA (cDNA/NTPy/rGO/Au), the presence of which, in addition to amounts of ethanolamine, paused this self-organizing stage and updated the readouts to 64 mdeg (Figure 2A, line d).
[Figure 2]
To highlight such cDNA/NTPy/rGO regime, a paralleled comparison was undertaken. In one hand, the SPR measurement was switched to the angle-change mode, for 1 μM tDNA (see Supporting Information: 5. DNA Hybridization for Enzyme-Linked Oligonucleotide Sorbent Assay), the calculation of which equals 189 mdeg (Figure 2B, line a). Referring to the SPR inferential statistics (Schasfoort and Tudos, 2008), the resultant density of tDNA on behalf of dsDNA on chip was estimated to be 1.93×10−4 nmol·mm−2. In another hand, 3-mercaptopropionic acid (MPA)-assembled exposure was directly subjected to covalent amidation with aminated cDNA. On the contrary to its counterpart, a less distinct shift of angles was attained (85 mdeg, e.g. 8.87×10−5 nmol·mm−2 tDNA) (Figure 2B, line b), which corroborated that the schematic of Figure 2B, line a exceled the one of line b at a virtually 200% apparent concentration, an obvious contribution from NTPy/rGO. What is more, a trial in adapting tDNA/NTPy/rGO for the control of cDNA/NTPy/rGO was intended. Unexpectedly, not even a legible swing of angle has exhibited (Figure 2B, line c), demonstrating 8
excellent anti-interfere ability of NTPy/rGO with no regards to the nonspecific issue. One more thing, to clarify the privilege of NTPy/rGO matrices, the hybridization of tDNA with thiolated cDNA monolayer upon gold plate was also conducted (Figure 2B, line d), the outcome of which event (i.e. dsDNA/Au) turned out to be a displacement (60 mdeg) slightly less than one-third of that in line a. Last but not the least, to preclude the false-positive arisen by HRP-SA, another control was practised and interpreted as Figure 2B, line e that non-specific binding of HRP-SA could hardly contract our system. Admittedly from this angle, NTPy encompassed somewhat a non-fouling surface. In a nutshell, this remarkable SPR sensitization was not only rooted in the marvelous quantum electrodynamic characteristics of rGO (Kuzmin, et al., 2014; Ferrari and Basko, 2013), but also benefitted from the NTPy-guided assembling and array of cDNA, which serves literally as a top priority in bio-inorganic interfacing engineering. 3.3. AFM revelation of noncovalent assembly amid dsDNA/NTPy/rGO Although FESEM and (E)SPR were capable of silhouetting rGO and HRP-SA vividly (vide infra); nevertheless, when getting down to those intangible molecular interactions, neither will come in handy except for atomic force microscopy (AFM). Basically, in a scanning area of 2×2 μm, the 3D topological graph of rGO maintained a fragmented outline (Figure 3A), identical to Figure 1D, the pieces of which occupied almost the entire region with a broad size distribution from nano- to micro-meters, while the average in-plane height fixed at about 2 nm (Z-offset = 0 nm). This conveys a notion that the electro- reduction actually generated nanosheets, not atomic monolayered graphene. As for the in situ adsorption of NTPy onto rGO, given the van der Waals radius of NTA, the general altitude of sampled sector was upraised by ~6 nm with sparsely scattered white ″needles″ in the vicinity (Figure 3B), 9
convincing us the existence of rGO-supported NTPy, sitting tight and secure via π-conjugation. Since the applied quantity of tDNA was always inferior to that of cDNA, no wonder the circumstances before and after hybridization could be merged and discriminated in the same image as Figure 3C, which as ever managed a partially blurry and rough contour of rGO beneath cDNA/NTPy. Considering the concept of double helix (dsDNA)(Mata and Luedtke, 2015), tens of separately vertical cones and pyramids, tall and short, could be distinguished as dsDNA/NTPy/rGO and cDNA/NTPy/rGO, repectively. In summary, this topographic mapping not only expounded the mathematic evaluation that NTPy/rGO offered a powerful scaffold for improving probe fittings, but promised the feasibility of our devised method.
[Figure 3]
3.4. Upside labeling and biocatalytic polymerization for DNA analysis Employing tDNA to metaphorize a bridge joining two partitions, since the problem of ″lower-half″ solved, as a fair consideration, attentions hereto was paid to the upper part (see Supporting Information: 6. Biocatalytic Polymerization-Triggered Mass Effect
of
Tracers),
which
was
to
be
elucidated
in
Figure
4A.
The
cDNA/NTPy/rGO/Au as the starting point presented a steady-state SPR angle of 77 mdeg (Figure 4A, black line a). As soon as 0.1 nM tDNA were anchored, the angular readings increased to 105 mdeg at its equilibrium (Figure 4A, black line b), right behind which the trajectory began to soar as long as the continuous incubation of 1 μM rDNA (123 mdeg, Figure 4A, black line c). Imminently, HRP-SA fell upon the exposed terminal of rDNA, revealing a 235-mdeg segment (Figure 4A, black line d); and straight away catalyzed the proton relay between aniline and H2O2. 10
Instantaneously, the black line d was outweighed by the emergence of PANi (435 mdeg, Figure 4A, black line e). Being aware of that both cDNA and rDNA held the biotin ligand, which would presume that rDNA might compete NTA−Ni2+ with cDNA or possibly cDNA would bind with HRP-SA not NTA−Ni2+. To neutralize these complications, control experimentation (the red line in Figure 4A) was conducted; yet one could claim from the insignificant yield - a negative result even if any polymerization involved, that without tDNA, there would be no such concerned crosstalk issues. To sum up, phase d and e gained a total net weight of 200 mdeg, merely by one seventh of which was deviated from section c to b (28 mdeg). As a matter of fact, this discrepancy in sensorgram underscored a greatly efficient redox-induced polymerization, which was tailored later on towards a sensitive DNA biosensor.
[Figure 4]
Analogous to Figure 1B, ESPR was exercised to validate the polymerization of aniline. As default, the profile of HRP-SA/rDNA/dsDNA/NTPy/rGO in acetate buffer was transplaced to 0 mdeg (Figure 4B, line a). Aniline initiated to be oxidized by H2O2 altogether with the coexistent HRP, which in return begot an in situ selfpolymerization into PANi and productively a tremendous SPR reply. Eliminating excessive aniline and H2O2 by HAc/NaAc, an overhead flatline was reached (Figure 4B, line b), a dead-stop end-point to the reaction. This low- to high-profile transcendence illuminated that PANi sedimented spontaneously, the electroactivity of which and its notable impact on SPR replication were magnified in Figure 4B, inset. Meanwhile, CV as the driving force was compiled in Figure 4C. For 11
HRP-SA/rDNA/dsDNA/NTPy/rGO, without aniline, no peaks stuck out in the potential between 0.4 and −0.3 V (Figure 4C, line a), which coincided with the situation of Figure 4B, line a. In stark contrast, a well-defined pair of quasi-reversible peaks occurred at 0.17 and 0.086 V (Figure 4C, line b), and concurrently for Figure 3B, line b, only if aniline was timed to be incorporated. The phenomenon bears resemblance with the published papers (Lai, et al., 2014; Peng, et al., 2010; Sheng, et al., 2011), which substantiated the successful coagulation of PANi by catalysis of enzymes. To deliver an absolute justification for the deduced mechanism, FESEM was taken resort to excavate the visual truth of enzymolytic polymerization (Figure 4D). Attributed to NTPy/rGO, cDNA is eligible to distribute uniformly, which could be projected indirectly at such resolution from thickly dotted infinitesimal proteins (Figure 4D, picture a). Cramming numerous hotspots into a limited space, just imaging the tiny SPR cavity, would definitely cause the initiator: aniline and H2O2, to converge and react collectively and fiercely. In consequence, filamentous flocculation featured in Figure 4D, picture b, which was designated undoubtedly as the PANi nanofibers (Wang, et al., 2012; Wei, et al., 2011). In a word, on the account of preceding logistical reasoning, it is confirmed that the on-spot biocatalytic polymerization of aniline indeed happened and behaved quite prominent by simple manual control, which is briefed in a formula below: Aniline + H2O2 → PANi↓ + H2O
(HRP-SA/rDNA/dsDNA/NTPy/rGO/Au)
Now, this strategy was ready to synergize with the verified advantages of NTPy/rGO for a coalescent SPR enhancement. 3.5. Analytical performance, uniformity and specificity Making full use of the developed platform, the kinetic records of tDNA with 12
concentrations from 10 fM to 1.0 nM were enumerated in Figure 5A, inset, the logarithmic values of which was proportional to their corresponding angular shifts. A calibration curve was fitted: y = 99.66·logx + 63.72 with a good linearity (regression coefficient: 0.997) over four orders of magnitudes (Figure 5A). Every data point has been duplicated for at least three times. The intra-assay and inter-assay precisions of the assay were examined by detecting 1.0 pM tDNA. The relative standard deviation (RSD) for measurements at five different spots on one SPR chip was 8.8%, while RSD for five parallel measurements on five separate chips 8.5%, indicating good accuracy and acceptable fabrication reproducibility. Ten measurements of SPR signal at 1.0 pM tDNA upon continuous channeling showed excellent reliability and stability (RSD = 2.0%). To assess the detection limit, a blank was defined beforehand denoting null tDNA, whereas aniline could still be converted into PANi in the presence of HRP, which meant the zero-indicator was mainly derived from the additives: aniline and H2O2. At this condition, a lower and upper detection limit of 7.91 fM and ~10 nM (S/N = 3) was acquired, respectively, which surpassed those of most recent DNA sensors (Degliangeli, et al., 2014; Guo, et al., 2014; Qiu, et al., 2015; Song, et al., 2015), proving the importance of synergistic sensitization.
[Figure 5]
The specificity of this assay was examined by normalizing the SPR intensities among four sequences, namely, tDNA and, 1-Base (1B), 2-Base (2B) and 3-Base (3B) mismatches at an equivalent 0.1 nM, which, in the order of B1, B2 and B3, were computed to be 8.1, 5.5 and 7.0%, respectively, of that of the perfectly 13
complementary (Figure 5B). This setup of highly-ordered recognizing matrix on superflat graphene sheet maximized hybridizing efficacy of tDNA, while suppressing the opportunity for mismatch expressions, thus ensured an acceptable specificity for the possible genotyping of single-nucleotide polymorphism.
4. Conclusion
A dual-multiplier sensitized SPR-DNA sensing was invented following a bidirectional form-logic ideology: (1) Bottom-up, in situ electro-reduced GO was endeavored to support NTPr as quality scaffolds for overcoming bio-inorganic interfacing issues, leading to an enhancement factor by 200% compared with the ordinary thiolated manner; and (2) Top-down, aniline was polymerized into PANi to incite mass-effect by on-line biocatalysis, resulting in a 6-time signal amplification beyond the classic label-free models. In this sense, by holing through signal transduction at the baseboard to its up-stream initiation and cascading amplification, an ultrasensitive determination of femtomolar-level nucleic acid was harvested. This well-designed sensor building could be expanded to tap the potency of SPR universality in relevant fields, such as early-cancer diagnoses.
Acknowledgements
This research was supported by National Natural Science Foundation of China (Grant No.21175114, 21305067 and 61371039), Natural Science Foundation of Jiangsu Province (BK20130754), Ph.D. Fund of Ministry Of Education for Young Teachers (0133219120019), the Fundamental Research Funds for the Central Universities (30920140112009, 30916011204), and ″A Project Funded by the Priority
14
Academic Program Development of Jiangsu Higher Education Institutions (PAPD)″.
References Bao, Q.; Loh, K. P. 2012. ACS Nano. 6, 3677−3694. Baur, J., Holzinger, M., Gondran, C., Cosnier, S. 2010. Electrochem. Commun. 12, 1287−1290. Bonaccorso, F., Sun, Z., Hasan, T., Ferrari, A. C. 2010. Nat. Photonics. 4, 611−622. Cai, B., Huang, L., Zhang, H., Sun, Z., Zhang, Z., Zhang, G. J. 2015. Biosens. Bioelectron. 74, 329−334. Camposeo, A., Persano, L., Manco, R., Wang, Y., Carro, P. D., Zhang, C., Li, Z. Y., Pisignano, D., Xia, Y. 2015. ACS Nano. 9, 10047−10054. Chen, F., Surkus, A. E., He, L., Pohl, M. M., Radnik, J., Topf, C., Junge, K., Beller, M. 2015. J. Am. Chem. Soc. 137, 11718−11724. Cheng, Z., Wang, Z., Gillespie, D. E., Lausted, C., Zheng, Z., Yang, M., Zhu, J. 2015. Anal. Chem. 87, 1466−1469. Cibulskis, K.; Lawrence, M. S.; Carter, S. L.; Sivachenko, A.; Jaffe, D.; Sougnez, C.; Gabriel, S.; Meyerson, M.; Lander, E. S.; Getz, G. 2013. Nat. Biotechnol. 31, 213−219. Cosnier, S., Holzinger, M. 2011. Chem. Soc. Rev. 40, 2146−2156. Das, M. R., Sarma, R. K., Saikia, R., Kale, V. S., Shelke, M. V.,Sengupta, P. 2011. Coloids and Surfaces B: Biointerfaces. 86, 16−22. Degliangeli, F., Kshirsagar, P., Brunetti, V., Pompa, P. P., Fiammengo, R. 2014. J. Am. Chem. Soc. 136, 2264−2267. DeKosky, B. J., Ippolito, G. C., Deschner, R. P., Lavinder, J. J., Wine, Y., Rawlings, B. M., Varadarajan, N., Giesecke, C., Dorner, T., Andrews, S. F. 2013, Nat. Biotechnol. 15
31, 8 166−169. Ding, X., Liow, C. H., Zhang, M., Huang, R., Li, C., Shen, H., Liu, M., Zou, Y., Gao, N., Zhang, Z., Li, Y., Wang, Q., Li, S., Jiang, J. 2014. J. Am. Chem. Soc. 136, 15684−15693. Eigler, S., Hirsch, A. 2014. Angew. Chem. Int. Ed. 53, 7720−7738. Fang, Y., Chen, S., Wang, W., Shan, X., Tao, N. 2015. Angew. Chem. Int. Ed. 54, 2538−2542. Ferrari, A. C., Basko, D. M. 2013. Nat. Nano. 8, 235−246. Geldmeier, J., König, T., Mahmoud, M. A., El-Sayed, M. A., Tsukruk, V. V. 2014. Adv. Funct. Mater. 24, 6797−6805. Guo, L., Xu, Y., Ferhan, A. R., Chen, G., Kim, D. H. 2013. J. Am. Chem. Soc. 135, 12338−12345. Guo, S., Yang, F., Zhang, Y. L., Ning, Y., Yao, Q. F., Zhang, G. J. 2014. Anal. Methods. 6, 3598−3603. Guo, X. J. 2012. Biophotonics. 5, 483−501. Han, J., Sa, Y. J., Shim, Y., Choi, M., Park, N., Joo, S. H., Park, S. 2015. Angew. Chem. Int. Ed. 54, 12622−12626. Harrison, K. L., Biedermann, L. B., Zavadil, K. R. 2015. Langmuir. 31, 9825−9832. Hou, J., Zheng, Y., Su, Y., Zhang, W., Hoshide, T., Xia, F., Jie, J., Li, Q., Zhao, Z., Ma, R., Sasaki, T., Geng, F. 2015. J. Am. Chem. Soc. 137, 13200−13208. Jiang, N. N., Shao, L., Wang, J. F. 2014. Adv. Mater. 26, 3282−3289. Jimenez-Monroy, K. L., Kick, A., Eersels, K., Grinsven, B., Wagner, P., Mertig, M. 2013. Phys. Status Solidi A 210. 5, 918−925. Krishnan, S., Mani, V., Wasalathanthri, D., Kumar, C. V., Rusling, J. F. 2011. Angew. Chem. Int. Ed. 50, 1175−1178. 16
Kundu, A., Layek, R. K., Kuila, A., Nandi, A. K. 2012. ACS Appl. Mater. Interfaces. 4, 5576−5582. Kumar, V. D. A., Kumar, V. D. A., Malathi, S., Jagaeedesh, P. 2014. Adv. Mater. Res. 984, 1345−1349. Kuzmin, D. A., Bychkov, I. V., Shavrov, V. G. 2014. Photonics and Nanostructures − Fundamentals and Applications. 12, 473−481. Lai, G., Zhang, H., Tamanna, T., Yu, A. 2014. Anal. Chem. 86, 1789−1793. Li, X., Wang, Y., Wang, L., Wei, Q. 2014. Chem. Commun. 50, 5049−5052. Liu, J., Zhao, Q., Liu, J. L., Wu, Y. S., Cheng, Y., Ji, M. W., Qian, H. M., Hao, W. C., Liu, C., Cui, D., Li, H. 2010. Biosens. Bioelectron. 26, 255−261. Liu, X., Qian, T., Na Xu, N., Zhou, J., Guo, J., Yan. C. 2015. Carbon. 92, 348−353. Losurdo, M., Bergmair, I., Dastmalchi, B., Kim, T. H., Giangregroio, M. M., Jiao, W., Bianco, G. V., Brown, A. S., Hingerl, K., Bruno, G. 2014. Adv. Funct. Mater. 24, 1864−1878. Mata, G., Luedtke, N. W. 2015. J. Am. Chem. Soc. 137, 699−707. Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E. B., Doudna, J. A., Liu, D. R. 2013. Nat. Biotechnol. 31, 839−843. Pei, H., Lu, N., Wen, Y., Song, S., Liu, Y., Yan, H., Fan, C. 2010. Adv. Mater. 22, 4754−4758. Peng, Y. F., Yi, G. S., Gao, Z. Q. 2010. Chem. Commun. 46, 9131−9133. Pia, E. A. D., Martinez, K. L. 2015. PLoS One. DOI:10.1371/journal.pone.0124303. Prathap, M. U. A., Chaurasia, A. K., Sawant, S. N., Apte, S. K. 2012. Anal. Chem. 84, 6672−6678. Qiu, X., Liu, X., Zhang, W., Zhang, H., Jiang, T., Fan, D., Luo, Y. 2015. Anal. Chem. 87, 6303−6310. 17
Schasfoort, R. B. M., Tudos, A. J. 2008. Handbook of Surface Plasmon Resonance. Sheng, Q. L., Wang, M. Z., Zheng, J. B. 2011. Sens. Actuators. B. 160, 1070−1077. Song, C., Xie, G., Wang, L., Liu, L., Tian, G., Xiang, H. 2014. Biosens. Bioelectron. 58, 68−74. Song, C., Wang, G. Y., Kong, D. M. 2015. Biosens. Bioelectrons. 68, 239−244. Stern, L., Grajoer, M., Levy, U. 2014. Nat. Commun. 5, DOI: 10.1038/ncomms5865. Subramanian, P., Lesniewski, A., Kaminska, I., Vlandas, A., Vasilescu, A., Niedziolka-Jonsson, J., Pichonat, E., Happy, H., Boukherroub, R., Szunerits, S. 2013. Biosens. Bioelectron. 50, 239−243. Subramanian, P., Barka-Bouaifel, F., Bouckaert, J., Yamakawa, N., Boukerroub, R., Szunerits, S. 2014. ACS Appl. Mater. Interfaces. 6, 5422−5431. Tang, C., Paul, A., Alam, M. P., Roy, B., Wilson, W. D., Hecht, S. M. 2014. J. Am. Chem. Soc. 136, 13715−13726. Wang, F., Wang, B., Feng, L., Liu, L., Lv, F., Wang, Y., Wang, S. 2014. Angew. Chem. Int. Ed. 53, 424−428. Wang, L., Zhu, C., Han, L., Jin, L., Zhou, M., Dong, S. 2011. Chem. Commun. 47, 7794−7796. Wang, L., Wu, X. L., Xu, W. H., Huang, X. J., Liu, J. H., Xu, A. W. 2012. ACS Appl. Mater. Interfaces. 4, 2686−2692. Wang, W., Han, N., Li, R., Han, W., Zhang, X., Li, F. 2015. Anal. Chem. 87, 9302−9307. Wang, Z. G., Zhan, P. F., Ding, B. Q. 2013. ACS Nano. 7, 1591−1598. Wei, D., Li, H., Han, D., Zhang, Q., Niu, L., Yang, H., Bower, C., Andrew, P., Ryhanen, T. 2011. Nanotechnology. 22, 245702−245708. Wijaya, E., Lenaerts, C., Maricot, S., Hastanin, J., Habraken, S., Vilcot, J. P., 18
Boukherroub, R., Szunerits, S. 2011. Curr. Opin. Solid State Mater. Sci. 15, 208−224. Xiang, Q., Cheng, B., Yu, J. 2015. Angew. Chem. Int. Ed. 54, 11350−11366. Xu, Y., Li, K., Qin, W., Zhu, B., Zhou, Z., Shi, J., Wang, K., Hu, J., Fan, C., Li, D. 2015. Anal. Chem. 87, 1968−1973. Xue, T.; Cui, X.; Guan, W.; Wang, Q.; Liu, C.; Wang, H.; Qi, K.; Singh, D. J.;Zheng, W. 2014. Biosens. Bioelectron. 58, 374−379. Yan, H., Cheng, H., Yi, H., Lin, Y., Yao, T., Wang, C., Li, J., Wei, S., Lu, J. 2015. J. Am. Chem. Soc. 137, 10484−10487. Yang, H., Lin, C. W., Hua, M. Y., Liao, S. S., Chen, Y. T., Chen, H. C., Weng, W. H., Chuang, C. K., Pang, S. T., Ma. C. C. M. 2014. Adv. Mater. 26, 3662−3666. Yu, X., Li, Y., Wu, J., Ju, H. 2014a. Anal. Chem. 86, 4501−4507. Yu, X., Park, H. S. 2014b. Carbon. 77, 59−65. Yuan, P., Deng, S., Xin, P., Ji, X., Shan, D., Cosnier, S. 2015. Biosens. Bioelectron. 74, 183−189. Zagorodko, O., Spadavecchia, J., Serrano, A. Y., Larroulet, I., Pesquera, A., Zurutuza, A., Boukherroub, R., Szunerits, S. 2014. Anal. Chem. 86, 11211−11216. Zhang, Q., Xu, X., Li, H., Xiong, G., Han Hu, H., Fisher, T. S. 2015. Carbon. 93, 659−670. Zhang, Y., Bai, X., Wang, X., Shiu, K. K., Zhu, Y., Jiang, H. 2014. Anal. Chem. 86, 9459−9465. Zhu, N., Han, S., Gan, S., Ulstrup, J., Chi, Q. 2013. Adv. Funct. Mater. 23, 5297−5306.
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Figure 1 (A) CV (blue) and ESPR (red) responses during the eletrodeposition of GO, curve a: 1st and b: 500th cycle. (B) SPR plot in consistence with ESPR. (C) SPR signals of dsDNA/NTPy/rGO in a function of CV loops. (D) SEM images of bare Au (a) and rGO/Au (b) chips. Figure 2 (A) SPR baselines of bare Au (a), rGO/Au (b), NTPy/rGO/Au (c), and cDNA/NTPy/rGO/Au (d). (B) Real-time angle shifts of dsDNA/NTPy/rGO/Au (a), dsDNA/MPA/Au (b), bare Au (c), dsDNA/Au (d), and HRP-SA/NTPy/rGO/Au (e). The arrows and texts indicate where and how to proceed the stage of interest. Figure 3 AFM images of (A) rGO/Au, (B) NTPy/rGO/Au, and (C) dsDNA/NTPy/ rGO/Au. Figure 4 (A) Sampling points on real-time trajectory: cDNA/NTPy/rGO/Au (a), dsDNA/NTPy/rGO/Au (black) and cDNA/NTPy/rGO/Au+PBS (red) (b), rDNA/ dsDNA/NTPy/rGO/Au
(c),
HRP-SA/rDNA/
dsDNA/NTPy/rGO/Au
(d),
and
(d)+aniline/H2O2 (e). The arrows and texts indicate where and how to proceed the following stage. (B) SPR responses, (C) CVs and (D) SEM images before (a) and after (b) the polymerization of aniline. Inset B: zoom-in of (b). Figure 5 (A) The calibration curve (concentration of tDNA: 1.0 10.0, 100.0 fM, 1.0, 10.0, 100.0 pM, and 1.0 nM, from bottom to top). (B) Normalized SPR intensities of tDNA and, 1-, 2-, and 3-Base mismatches (0.1 nM). Inset: The corresponding SPR responses. Scheme 1 Schematic illustration of the noncovalent functionalization of rGO for sensitizing SPR-Based DNA sensing synergistically with biocatalytic polymerization.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
Highlights
An ultrasensitive SPR-based biosensing mode was developed with dualamplifiers incorporated within both the substrate and the tracing tag, which synergized altogether to accomplish a femtomolar-level DNA detection.
Nickel-chelated nitrilotriacetic pyrenes supported by electro-reduced graphene nanosheets were in situ self-assembled noncovalently on SPR chip for highly ordered and densely populated immobilization of capture probes.
Aniline was selected as a real-time initiator for biocatalytic polymerization by labeled enzymes. Its oxidative product of polyaniline triggered mass effect upon the above interface, jointly resulting in a significant sensitization on SPR output.
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PII: DOI: Reference:
S0956-5663(16)30659-5 http://dx.doi.org/10.1016/j.bios.2016.07.031 BIOS8912
To appear in: Biosensors and Bioelectronic Received date: 30 November 2015 Revised date: 30 June 2016 Accepted date: 7 July 2016 Cite this article as: Pei-Xin Yuan, Sheng-Yuan Deng, Chuan-Guang Yao, Ying Wan, Serge Cosnier and Dan Shan, Polymerization Amplified SPR−DNA Assay on Noncovalently Functionalized Graphene, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.07.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Polymerization Amplified SPR−DNA Assay on Noncovalently Functionalized Graphene Pei-Xin Yuana, Sheng-Yuan Denga,*, Chuan-Guang Yaoa, Ying Wan,b Serge Cosniera,c，Dan Shana,* a
Sino-French Laboratory of Biomaterials and Bioanalytical Chemistry, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China b Intelligent Microsystem Technology and Engineering Center, School of Mechanical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China c University of Grenoble, Alpes−CNRS, DCM UMR 5250, F−38000 Grenoble, France
[email protected] [email protected] *
Corresponding author. Tel./Fax: +86−(25)−84303107
Abstract A highly efficient surface plasmon resonance (SPR)-based DNA assay was developed, by employing noncovalently functionalized graphene nanosheets as a substrate, and enzymatic catalysis-induced polymerization as mass relay. The objective of this strategy was manifold: first of all, to sensitize the overall SPR output by in situ optimized electrogeneration of graphene thin-film, which was characterized by atomic force microscopic topography; secondly, to regulate the self-assembly and orientation of biotinylated capture probes on nickel-chelated nitrilotriacetic acid (NTA) scaffolds, that anchored onto graphene-supported pyrenyl derivatives; and lastly, to synergize the signal amplification via real-time conversion of the additive aniline into polyaniline precipitation by horseradish peroxidase-tagged reporters. With this setup, a precise and replicable DNA sensing platform for specific targets was achieved with a detection limit down to femtomolar, thus demonstrating a beneficial exploration and 1
exploitation of two-dimensional nanomaterials as unique SPR infrastructure. The possibility of such ″bottom-up″ architecture mounted with ″top-down″ weight reactor would be most likely extensible and adaptable to protein determinations.
Keywords: surface plasmon resonance; DNA assay; electrochemically reduced graphene nanosheets; pyrene-tethered nitrilotriacetic acid; biocatalytic polymerization
1. Introduction
Owing to its intrinsic capability of interpreting near-interface heterogeneity, surface plasmon resonance (SPR) as an established analytical technology, has predominated in the study of ″bio-mass″ interaction and migration (Fang, et al., 2015; Stern, et al., 2014; Wijaya, et al., 2011). Comprehensively, dual aspects could be well exemplified in the manipulation of sequence-dependent DNA hybridization, which, in its very essence, embodies an immovable code for versatile utilizations on SPR platform, or more pertinently, the DNA biosensing (Cibulskis, et al., 2013; DeKosky, et al., 2013; Pattanayak, et al., 2013). Theoretically, oscillatory frequency, dielectric constant and proximal thickness comprise the primary variables for SPR signal transduction (Guo, 2012); while in realistic practices, far-end modulation towards the sensitization of SPR response became a strategic preference in terms of either composited probes as nano-resonators (Ding, et al., 2014; Krishnan, et al., 2011), or molecular biology, typically the terminal deoxynucleotidyl transferase extension, rolling cycle amplification, and polymerase chain reaction (Li, et al., 2011; Yuan, et al., 2015). On the other side, none could deny the fact that the load and layout of underlying capture units exert a most neglected profound influence on the explicit efficiency of standard 2
protocols (Cosnier and Holzinger, 2011; Guo, et al., 2013), where the dilemma also hides that the valid density of immobilization turned out below consensus by the plausible Au−S bonding (Jimenez-Monroy, et al., 2013; Tang, et al., 2014) on conventional SPR chips (Cai, et al., 2015; Song, et al., 2014). To circumvent such predicament, tectonically by observing the bottom-up development, reconstituting fine structures of the surface was motivated in recent years (Liu, et al., 2015; Subramanian, et al., 2014; Zagorodko, et al., 2014), infusing vigor and vitality in the fundamentals of SPR-centered bioanalysis. For instance, the transplant of tetrahedral DNA origami individuals accomplished an extraordinarily non-blocking, mechanically rigid, yet epitaxially flexible substrate (Pei, et al., 2010), particularly qualified for biometric identification (Kumar, et al., 2014). By contrast, in a holistic and macroscopic view, it is universally acknowledged that on-site large-scale overlay, by electrospun fibration (Camposeo, et al., 2015), LangmuirBlodgett filming (Geldmeier, et al., 2014), or chemical vapor deposition (Liu, et al., 2010; Losurdo, et al., 2014), emerged to be a relatively and increasingly more accessible and accountable approach. Therein, an incarnation of SPR immunoassay has been dedicated by virtue of polymethyl-methacrylate-assisted transfer of graphene-like two-dimensional nanomaterials (Chen, et al., 2015; Han, et al., 2015; Hou, et al., 2015; Xiang, et al., 2015; Yan, et al., 2015), precisely due to their enormously lamellar flatness and periodically planar homogeneity (Das, et al., 2011; Subramanian, et al., 2013). On the merit of these potent properties, decoration of densely populated gold nanoparticles (Xue, et al., 2014; Yang, et al., 2014; Zhang, et al., 2014; Zhu, et al., 2013), and noncovalent self-assembly of highly ordered scaffolds (Eigler and Hirsch, 2014), as fruitful measures for post-functionalization, further achieved unprecedented SPR enhancement in DNA and protein detections 3
(Wang, et al., 2015; Zagorodko, et al., 2014), which shall be retrospectively ascribed to the matter of frameworks at bottom. Hierarchically, this non-routine perspective could be assumed compatible and even able to synergize cohesively with rational top-level designs, hence consummating an ultimate pattern of SPR-based methodologies. Inspired by the above heuristic review and progressive insight, a tentative research focusing on the SPR reinforcement at both ends for the determination of oligonucleotides was proposed (Scheme 1), which commenced with in situ electrophoretic precipitation of graphene oxide (GO) in aqueous solution to a membrane of graphene nanosheets (rGO) as capacious infrastructure. As a preliminary, cyclic voltammogram (CV)-tandem SPR (ESPR) proceeded to depict simultaneously this facile modification; whereas microscopic imaging illustrated the stepwise upward constructions. To accommodate DNA probes at its best, also to regulate their alignment, abundant nickel-chelated nitrilotriacetic acids (NTA) were tethered onto rGO-supported pyrene groups (NTPy). This scaffolding fabric, with reported association (kon = (1.1 ± 0.5) × 106 / Ms) and dissociation (koff = (7.1 ± 1.1) × 10−4 / s) constants comparable to the bindings of streptavidin/biotin (Pia and Martinez, 2015), circumscribed the on-chip orientation and footholds allowed for only biotinylated biomolecules (cDNA) via axial ligation of Ni2+−S (nickel perchlorate or Ni(ClO4)2−biotin), rendering rather vast solid-state expression of targets (tDNA) than that on traditional monolayer of Au−thiols. Moreover, for the sake of stoichiometric cascade, a sandwich format was implemented stereochemically to bridge the capture with the bio-reporter (rDNA): horseradish peroxidase-labeled streptavidin (HRP-SA), enabling a dynamic biocatalytic polymerization of aniline the reductant into polyaniline (PANi) (Jiang, et al., 2014; Wang, et al., 2013; Xu, et al., 2015). As a 4
conductive polymer (Prathap, et al., 2012; Yu, et al., 2014a), its long-chains sank and intertwined gradually, overweighing its carrier and accordingly disturbing specific conductance in the neighborhood. Therefore, an ultrasensitive DNA sensor came into being by coupling double SPR-multipliers in altitudinal series. In this way, such integration of reform about pristine matrices with joint mass effects by trigger would symbolize a navigable highway towards the uncharted horizon of SPR-related bioapplications.
[Scheme 1]
2. Experimental section
Please consult Supporting Information for elaborations on catalogued experimentation and instrumentation, concerning: 1. Materials; 2. Measurements and Apparatus; 3. Electrodeposition of Graphene Nanosheets; 4. NTA-Intermediated Immobilization of cDNA on rGO Surface; 5. DNA Hybridization for Enzyme-Linked Oligonucleotide Sorbent Assay; and 6. Biocatalytic Polymerization-Triggered Mass Effect of Tracers. As a reminder, the flow injection was adopted as a primary sampling method for most situations throughout the overall protocol, while the static method was implemented exceptionally for the hybridization of tDNA segment.
3. Results and discussion
3.1. Characterization and optimization of rGO thin-film As elaborated before, the SPR output counts substantially on the volume of probes.
5
In view of that, rGO was selected, as a capping agent on the whole, to veil the ready-made golden face. First and foremost, this originated from its comparatively immense surface-to-volume ratio than that of the bare one (Wang, et al., 2011). More than that, the basal plane of this browny shroud appeared to be fairly susceptible of ″stains″ at molecular level (Subramanian, et al., 2013). And remarkably, it was testified that the graphene-plated gold-leaf rectifies the power streams across dielectric sub-coatings within and redirects the field of polaritons (Bao and Lok, 2012; Bonaccorso, et al., 2010). These structure-activity relationships stimulated the exploitation of rGO in SPR, which was fulfilled facilely by CV (see Supporting Information: 3. Electrodeposition of Graphene Nanosheets). As shown in Figure 1A, blue curves, two reducing peaks rose with approximate onsets at −0.75 and 0.1 V, respectively, whose corresponding currents escalated with the incremental circulation (Figure 1A, curve a: 1st and b: 500th loop), suggesting an inherent structural evolution of GO. That is, the successive decarboxylation at negative ranges, during which the suspended GO platelets accumulated in the diffusion layer and adsorbed onto the electrode because of depolarization and hydrophobicity (Harrison, et al., 2015; Zhang, et al., 2015). This kinetics was reflected in a frequency (ω, i.e. ΔAngle) vs. potential diagram (Figure 1A, red curves), where conspicuous SPR angles fluctuated drastically, echoing against the voltammetric behaviors around that sole redox pair at a formal potential of 0.18 V. Coincidentally, both galvanic and ESPR responses, whether belonging to summits or baselines, augmented globally (Figure 1A, curves a and b). Especially, the inflation in charging currents alongside the loops indicated the ongoing thickening of deposited rGO flakes (Liu, et al., 2015; Subramanian, et al., 2011). Once investigated in details, ESPR plots in Figure 1A was then translated into a time-elapsed procession of Figure 1B, from which one could witness the significant 6
effect of incessantly iterative oxidation-reduction of GO on the SPR reflectivity; otherwise it would be overloaded in the long haul. In this case, the thickness of rGO must be optimized in light of the cycles in question, which was interrogated by comparing feedbacks among prefactoring dsDNA/NTPy/rGO (past various depositing intervals, dsDNA: cDNA+tDNA). As a result, the SPR responses for tDNA hybridization surged in company with the prolongation of electro-precipitation, and climbed to the climax at 500 repetitions (Figure 1C), manifesting the attachment of rGO did favor the habitats for cDNA up to its saturation. Hereafter, the signal collapsed probably out of a side-effect that the transmittance of rGO tier grew too low for the incident light to penetrate. Thus, the number 500 was adopted as an appropriate value for counting down. As a supplementary, rGO film by this means of batch-production was visualized in field-emission scanning electron microscopic (FESEM) image (Figure 1D), where rGO fragmentation overlapped into a wrinkled landscape by thermodynamic perturbation (Kundu, et al., 2012; Yu and Park, 2014b), however, still possessing velvety texture, a superior edge for more binding opportunities over pure gold.
[Figure 1]
3.2. Downside fabrication of cDNA/NTPy/rGO and its hybridizing capacity The settling of cDNA in the conceived scenario (see Supporting Information: 4. NTA-Intermediated Immobilization of cDNA on rGO Surface) was demonstrated in Figure 2A. Brand-new gold disk displayed a basic blank of −1630 mdeg (m°) in ultrapure water (Figure 2A, line a). The coverage of rGO raised the SPR angle up to −815 mdeg (Figure 2A, line b), a clear evidence of the rGO/Au outcome. Furthermore, 7
the baseline of rGO/Au was lifted up to a new plateau at −375 mdeg by the addition of NTPy (NTPy/rGO/Au)(Figure 2A, line c), implying that the pyrene species in NTPy had adhered to the hexagonal lattices of graphene via noncovalent π-π stacking (Eigler, et al., 2014). In such occasion, the entry of Ni2+ would naturally register itself a guest-host inclusion by peripheral carboxyls of NTA for an upright direction of follow-up occupants (Baur, et al., 2010): the cDNA (cDNA/NTPy/rGO/Au), the presence of which, in addition to amounts of ethanolamine, paused this self-organizing stage and updated the readouts to 64 mdeg (Figure 2A, line d).
[Figure 2]
To highlight such cDNA/NTPy/rGO regime, a paralleled comparison was undertaken. In one hand, the SPR measurement was switched to the angle-change mode, for 1 μM tDNA (see Supporting Information: 5. DNA Hybridization for Enzyme-Linked Oligonucleotide Sorbent Assay), the calculation of which equals 189 mdeg (Figure 2B, line a). Referring to the SPR inferential statistics (Schasfoort and Tudos, 2008), the resultant density of tDNA on behalf of dsDNA on chip was estimated to be 1.93×10−4 nmol·mm−2. In another hand, 3-mercaptopropionic acid (MPA)-assembled exposure was directly subjected to covalent amidation with aminated cDNA. On the contrary to its counterpart, a less distinct shift of angles was attained (85 mdeg, e.g. 8.87×10−5 nmol·mm−2 tDNA) (Figure 2B, line b), which corroborated that the schematic of Figure 2B, line a exceled the one of line b at a virtually 200% apparent concentration, an obvious contribution from NTPy/rGO. What is more, a trial in adapting tDNA/NTPy/rGO for the control of cDNA/NTPy/rGO was intended. Unexpectedly, not even a legible swing of angle has exhibited (Figure 2B, line c), demonstrating 8
excellent anti-interfere ability of NTPy/rGO with no regards to the nonspecific issue. One more thing, to clarify the privilege of NTPy/rGO matrices, the hybridization of tDNA with thiolated cDNA monolayer upon gold plate was also conducted (Figure 2B, line d), the outcome of which event (i.e. dsDNA/Au) turned out to be a displacement (60 mdeg) slightly less than one-third of that in line a. Last but not the least, to preclude the false-positive arisen by HRP-SA, another control was practised and interpreted as Figure 2B, line e that non-specific binding of HRP-SA could hardly contract our system. Admittedly from this angle, NTPy encompassed somewhat a non-fouling surface. In a nutshell, this remarkable SPR sensitization was not only rooted in the marvelous quantum electrodynamic characteristics of rGO (Kuzmin, et al., 2014; Ferrari and Basko, 2013), but also benefitted from the NTPy-guided assembling and array of cDNA, which serves literally as a top priority in bio-inorganic interfacing engineering. 3.3. AFM revelation of noncovalent assembly amid dsDNA/NTPy/rGO Although FESEM and (E)SPR were capable of silhouetting rGO and HRP-SA vividly (vide infra); nevertheless, when getting down to those intangible molecular interactions, neither will come in handy except for atomic force microscopy (AFM). Basically, in a scanning area of 2×2 μm, the 3D topological graph of rGO maintained a fragmented outline (Figure 3A), identical to Figure 1D, the pieces of which occupied almost the entire region with a broad size distribution from nano- to micro-meters, while the average in-plane height fixed at about 2 nm (Z-offset = 0 nm). This conveys a notion that the electro- reduction actually generated nanosheets, not atomic monolayered graphene. As for the in situ adsorption of NTPy onto rGO, given the van der Waals radius of NTA, the general altitude of sampled sector was upraised by ~6 nm with sparsely scattered white ″needles″ in the vicinity (Figure 3B), 9
convincing us the existence of rGO-supported NTPy, sitting tight and secure via π-conjugation. Since the applied quantity of tDNA was always inferior to that of cDNA, no wonder the circumstances before and after hybridization could be merged and discriminated in the same image as Figure 3C, which as ever managed a partially blurry and rough contour of rGO beneath cDNA/NTPy. Considering the concept of double helix (dsDNA)(Mata and Luedtke, 2015), tens of separately vertical cones and pyramids, tall and short, could be distinguished as dsDNA/NTPy/rGO and cDNA/NTPy/rGO, repectively. In summary, this topographic mapping not only expounded the mathematic evaluation that NTPy/rGO offered a powerful scaffold for improving probe fittings, but promised the feasibility of our devised method.
[Figure 3]
3.4. Upside labeling and biocatalytic polymerization for DNA analysis Employing tDNA to metaphorize a bridge joining two partitions, since the problem of ″lower-half″ solved, as a fair consideration, attentions hereto was paid to the upper part (see Supporting Information: 6. Biocatalytic Polymerization-Triggered Mass Effect
of
Tracers),
which
was
to
be
elucidated
in
Figure
4A.
The
cDNA/NTPy/rGO/Au as the starting point presented a steady-state SPR angle of 77 mdeg (Figure 4A, black line a). As soon as 0.1 nM tDNA were anchored, the angular readings increased to 105 mdeg at its equilibrium (Figure 4A, black line b), right behind which the trajectory began to soar as long as the continuous incubation of 1 μM rDNA (123 mdeg, Figure 4A, black line c). Imminently, HRP-SA fell upon the exposed terminal of rDNA, revealing a 235-mdeg segment (Figure 4A, black line d); and straight away catalyzed the proton relay between aniline and H2O2. 10
Instantaneously, the black line d was outweighed by the emergence of PANi (435 mdeg, Figure 4A, black line e). Being aware of that both cDNA and rDNA held the biotin ligand, which would presume that rDNA might compete NTA−Ni2+ with cDNA or possibly cDNA would bind with HRP-SA not NTA−Ni2+. To neutralize these complications, control experimentation (the red line in Figure 4A) was conducted; yet one could claim from the insignificant yield - a negative result even if any polymerization involved, that without tDNA, there would be no such concerned crosstalk issues. To sum up, phase d and e gained a total net weight of 200 mdeg, merely by one seventh of which was deviated from section c to b (28 mdeg). As a matter of fact, this discrepancy in sensorgram underscored a greatly efficient redox-induced polymerization, which was tailored later on towards a sensitive DNA biosensor.
[Figure 4]
Analogous to Figure 1B, ESPR was exercised to validate the polymerization of aniline. As default, the profile of HRP-SA/rDNA/dsDNA/NTPy/rGO in acetate buffer was transplaced to 0 mdeg (Figure 4B, line a). Aniline initiated to be oxidized by H2O2 altogether with the coexistent HRP, which in return begot an in situ selfpolymerization into PANi and productively a tremendous SPR reply. Eliminating excessive aniline and H2O2 by HAc/NaAc, an overhead flatline was reached (Figure 4B, line b), a dead-stop end-point to the reaction. This low- to high-profile transcendence illuminated that PANi sedimented spontaneously, the electroactivity of which and its notable impact on SPR replication were magnified in Figure 4B, inset. Meanwhile, CV as the driving force was compiled in Figure 4C. For 11
HRP-SA/rDNA/dsDNA/NTPy/rGO, without aniline, no peaks stuck out in the potential between 0.4 and −0.3 V (Figure 4C, line a), which coincided with the situation of Figure 4B, line a. In stark contrast, a well-defined pair of quasi-reversible peaks occurred at 0.17 and 0.086 V (Figure 4C, line b), and concurrently for Figure 3B, line b, only if aniline was timed to be incorporated. The phenomenon bears resemblance with the published papers (Lai, et al., 2014; Peng, et al., 2010; Sheng, et al., 2011), which substantiated the successful coagulation of PANi by catalysis of enzymes. To deliver an absolute justification for the deduced mechanism, FESEM was taken resort to excavate the visual truth of enzymolytic polymerization (Figure 4D). Attributed to NTPy/rGO, cDNA is eligible to distribute uniformly, which could be projected indirectly at such resolution from thickly dotted infinitesimal proteins (Figure 4D, picture a). Cramming numerous hotspots into a limited space, just imaging the tiny SPR cavity, would definitely cause the initiator: aniline and H2O2, to converge and react collectively and fiercely. In consequence, filamentous flocculation featured in Figure 4D, picture b, which was designated undoubtedly as the PANi nanofibers (Wang, et al., 2012; Wei, et al., 2011). In a word, on the account of preceding logistical reasoning, it is confirmed that the on-spot biocatalytic polymerization of aniline indeed happened and behaved quite prominent by simple manual control, which is briefed in a formula below: Aniline + H2O2 → PANi↓ + H2O
(HRP-SA/rDNA/dsDNA/NTPy/rGO/Au)
Now, this strategy was ready to synergize with the verified advantages of NTPy/rGO for a coalescent SPR enhancement. 3.5. Analytical performance, uniformity and specificity Making full use of the developed platform, the kinetic records of tDNA with 12
concentrations from 10 fM to 1.0 nM were enumerated in Figure 5A, inset, the logarithmic values of which was proportional to their corresponding angular shifts. A calibration curve was fitted: y = 99.66·logx + 63.72 with a good linearity (regression coefficient: 0.997) over four orders of magnitudes (Figure 5A). Every data point has been duplicated for at least three times. The intra-assay and inter-assay precisions of the assay were examined by detecting 1.0 pM tDNA. The relative standard deviation (RSD) for measurements at five different spots on one SPR chip was 8.8%, while RSD for five parallel measurements on five separate chips 8.5%, indicating good accuracy and acceptable fabrication reproducibility. Ten measurements of SPR signal at 1.0 pM tDNA upon continuous channeling showed excellent reliability and stability (RSD = 2.0%). To assess the detection limit, a blank was defined beforehand denoting null tDNA, whereas aniline could still be converted into PANi in the presence of HRP, which meant the zero-indicator was mainly derived from the additives: aniline and H2O2. At this condition, a lower and upper detection limit of 7.91 fM and ~10 nM (S/N = 3) was acquired, respectively, which surpassed those of most recent DNA sensors (Degliangeli, et al., 2014; Guo, et al., 2014; Qiu, et al., 2015; Song, et al., 2015), proving the importance of synergistic sensitization.
[Figure 5]
The specificity of this assay was examined by normalizing the SPR intensities among four sequences, namely, tDNA and, 1-Base (1B), 2-Base (2B) and 3-Base (3B) mismatches at an equivalent 0.1 nM, which, in the order of B1, B2 and B3, were computed to be 8.1, 5.5 and 7.0%, respectively, of that of the perfectly 13
complementary (Figure 5B). This setup of highly-ordered recognizing matrix on superflat graphene sheet maximized hybridizing efficacy of tDNA, while suppressing the opportunity for mismatch expressions, thus ensured an acceptable specificity for the possible genotyping of single-nucleotide polymorphism.
4. Conclusion
A dual-multiplier sensitized SPR-DNA sensing was invented following a bidirectional form-logic ideology: (1) Bottom-up, in situ electro-reduced GO was endeavored to support NTPr as quality scaffolds for overcoming bio-inorganic interfacing issues, leading to an enhancement factor by 200% compared with the ordinary thiolated manner; and (2) Top-down, aniline was polymerized into PANi to incite mass-effect by on-line biocatalysis, resulting in a 6-time signal amplification beyond the classic label-free models. In this sense, by holing through signal transduction at the baseboard to its up-stream initiation and cascading amplification, an ultrasensitive determination of femtomolar-level nucleic acid was harvested. This well-designed sensor building could be expanded to tap the potency of SPR universality in relevant fields, such as early-cancer diagnoses.
Acknowledgements
This research was supported by National Natural Science Foundation of China (Grant No.21175114, 21305067 and 61371039), Natural Science Foundation of Jiangsu Province (BK20130754), Ph.D. Fund of Ministry Of Education for Young Teachers (0133219120019), the Fundamental Research Funds for the Central Universities (30920140112009, 30916011204), and ″A Project Funded by the Priority
14
Academic Program Development of Jiangsu Higher Education Institutions (PAPD)″.
References Bao, Q.; Loh, K. P. 2012. ACS Nano. 6, 3677−3694. Baur, J., Holzinger, M., Gondran, C., Cosnier, S. 2010. Electrochem. Commun. 12, 1287−1290. Bonaccorso, F., Sun, Z., Hasan, T., Ferrari, A. C. 2010. Nat. Photonics. 4, 611−622. Cai, B., Huang, L., Zhang, H., Sun, Z., Zhang, Z., Zhang, G. J. 2015. Biosens. Bioelectron. 74, 329−334. Camposeo, A., Persano, L., Manco, R., Wang, Y., Carro, P. D., Zhang, C., Li, Z. Y., Pisignano, D., Xia, Y. 2015. ACS Nano. 9, 10047−10054. Chen, F., Surkus, A. E., He, L., Pohl, M. M., Radnik, J., Topf, C., Junge, K., Beller, M. 2015. J. Am. Chem. Soc. 137, 11718−11724. Cheng, Z., Wang, Z., Gillespie, D. E., Lausted, C., Zheng, Z., Yang, M., Zhu, J. 2015. Anal. Chem. 87, 1466−1469. Cibulskis, K.; Lawrence, M. S.; Carter, S. L.; Sivachenko, A.; Jaffe, D.; Sougnez, C.; Gabriel, S.; Meyerson, M.; Lander, E. S.; Getz, G. 2013. Nat. Biotechnol. 31, 213−219. Cosnier, S., Holzinger, M. 2011. Chem. Soc. Rev. 40, 2146−2156. Das, M. R., Sarma, R. K., Saikia, R., Kale, V. S., Shelke, M. V.,Sengupta, P. 2011. Coloids and Surfaces B: Biointerfaces. 86, 16−22. Degliangeli, F., Kshirsagar, P., Brunetti, V., Pompa, P. P., Fiammengo, R. 2014. J. Am. Chem. Soc. 136, 2264−2267. DeKosky, B. J., Ippolito, G. C., Deschner, R. P., Lavinder, J. J., Wine, Y., Rawlings, B. M., Varadarajan, N., Giesecke, C., Dorner, T., Andrews, S. F. 2013, Nat. Biotechnol. 15
31, 8 166−169. Ding, X., Liow, C. H., Zhang, M., Huang, R., Li, C., Shen, H., Liu, M., Zou, Y., Gao, N., Zhang, Z., Li, Y., Wang, Q., Li, S., Jiang, J. 2014. J. Am. Chem. Soc. 136, 15684−15693. Eigler, S., Hirsch, A. 2014. Angew. Chem. Int. Ed. 53, 7720−7738. Fang, Y., Chen, S., Wang, W., Shan, X., Tao, N. 2015. Angew. Chem. Int. Ed. 54, 2538−2542. Ferrari, A. C., Basko, D. M. 2013. Nat. Nano. 8, 235−246. Geldmeier, J., König, T., Mahmoud, M. A., El-Sayed, M. A., Tsukruk, V. V. 2014. Adv. Funct. Mater. 24, 6797−6805. Guo, L., Xu, Y., Ferhan, A. R., Chen, G., Kim, D. H. 2013. J. Am. Chem. Soc. 135, 12338−12345. Guo, S., Yang, F., Zhang, Y. L., Ning, Y., Yao, Q. F., Zhang, G. J. 2014. Anal. Methods. 6, 3598−3603. Guo, X. J. 2012. Biophotonics. 5, 483−501. Han, J., Sa, Y. J., Shim, Y., Choi, M., Park, N., Joo, S. H., Park, S. 2015. Angew. Chem. Int. Ed. 54, 12622−12626. Harrison, K. L., Biedermann, L. B., Zavadil, K. R. 2015. Langmuir. 31, 9825−9832. Hou, J., Zheng, Y., Su, Y., Zhang, W., Hoshide, T., Xia, F., Jie, J., Li, Q., Zhao, Z., Ma, R., Sasaki, T., Geng, F. 2015. J. Am. Chem. Soc. 137, 13200−13208. Jiang, N. N., Shao, L., Wang, J. F. 2014. Adv. Mater. 26, 3282−3289. Jimenez-Monroy, K. L., Kick, A., Eersels, K., Grinsven, B., Wagner, P., Mertig, M. 2013. Phys. Status Solidi A 210. 5, 918−925. Krishnan, S., Mani, V., Wasalathanthri, D., Kumar, C. V., Rusling, J. F. 2011. Angew. Chem. Int. Ed. 50, 1175−1178. 16
Kundu, A., Layek, R. K., Kuila, A., Nandi, A. K. 2012. ACS Appl. Mater. Interfaces. 4, 5576−5582. Kumar, V. D. A., Kumar, V. D. A., Malathi, S., Jagaeedesh, P. 2014. Adv. Mater. Res. 984, 1345−1349. Kuzmin, D. A., Bychkov, I. V., Shavrov, V. G. 2014. Photonics and Nanostructures − Fundamentals and Applications. 12, 473−481. Lai, G., Zhang, H., Tamanna, T., Yu, A. 2014. Anal. Chem. 86, 1789−1793. Li, X., Wang, Y., Wang, L., Wei, Q. 2014. Chem. Commun. 50, 5049−5052. Liu, J., Zhao, Q., Liu, J. L., Wu, Y. S., Cheng, Y., Ji, M. W., Qian, H. M., Hao, W. C., Liu, C., Cui, D., Li, H. 2010. Biosens. Bioelectron. 26, 255−261. Liu, X., Qian, T., Na Xu, N., Zhou, J., Guo, J., Yan. C. 2015. Carbon. 92, 348−353. Losurdo, M., Bergmair, I., Dastmalchi, B., Kim, T. H., Giangregroio, M. M., Jiao, W., Bianco, G. V., Brown, A. S., Hingerl, K., Bruno, G. 2014. Adv. Funct. Mater. 24, 1864−1878. Mata, G., Luedtke, N. W. 2015. J. Am. Chem. Soc. 137, 699−707. Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E. B., Doudna, J. A., Liu, D. R. 2013. Nat. Biotechnol. 31, 839−843. Pei, H., Lu, N., Wen, Y., Song, S., Liu, Y., Yan, H., Fan, C. 2010. Adv. Mater. 22, 4754−4758. Peng, Y. F., Yi, G. S., Gao, Z. Q. 2010. Chem. Commun. 46, 9131−9133. Pia, E. A. D., Martinez, K. L. 2015. PLoS One. DOI:10.1371/journal.pone.0124303. Prathap, M. U. A., Chaurasia, A. K., Sawant, S. N., Apte, S. K. 2012. Anal. Chem. 84, 6672−6678. Qiu, X., Liu, X., Zhang, W., Zhang, H., Jiang, T., Fan, D., Luo, Y. 2015. Anal. Chem. 87, 6303−6310. 17
Schasfoort, R. B. M., Tudos, A. J. 2008. Handbook of Surface Plasmon Resonance. Sheng, Q. L., Wang, M. Z., Zheng, J. B. 2011. Sens. Actuators. B. 160, 1070−1077. Song, C., Xie, G., Wang, L., Liu, L., Tian, G., Xiang, H. 2014. Biosens. Bioelectron. 58, 68−74. Song, C., Wang, G. Y., Kong, D. M. 2015. Biosens. Bioelectrons. 68, 239−244. Stern, L., Grajoer, M., Levy, U. 2014. Nat. Commun. 5, DOI: 10.1038/ncomms5865. Subramanian, P., Lesniewski, A., Kaminska, I., Vlandas, A., Vasilescu, A., Niedziolka-Jonsson, J., Pichonat, E., Happy, H., Boukherroub, R., Szunerits, S. 2013. Biosens. Bioelectron. 50, 239−243. Subramanian, P., Barka-Bouaifel, F., Bouckaert, J., Yamakawa, N., Boukerroub, R., Szunerits, S. 2014. ACS Appl. Mater. Interfaces. 6, 5422−5431. Tang, C., Paul, A., Alam, M. P., Roy, B., Wilson, W. D., Hecht, S. M. 2014. J. Am. Chem. Soc. 136, 13715−13726. Wang, F., Wang, B., Feng, L., Liu, L., Lv, F., Wang, Y., Wang, S. 2014. Angew. Chem. Int. Ed. 53, 424−428. Wang, L., Zhu, C., Han, L., Jin, L., Zhou, M., Dong, S. 2011. Chem. Commun. 47, 7794−7796. Wang, L., Wu, X. L., Xu, W. H., Huang, X. J., Liu, J. H., Xu, A. W. 2012. ACS Appl. Mater. Interfaces. 4, 2686−2692. Wang, W., Han, N., Li, R., Han, W., Zhang, X., Li, F. 2015. Anal. Chem. 87, 9302−9307. Wang, Z. G., Zhan, P. F., Ding, B. Q. 2013. ACS Nano. 7, 1591−1598. Wei, D., Li, H., Han, D., Zhang, Q., Niu, L., Yang, H., Bower, C., Andrew, P., Ryhanen, T. 2011. Nanotechnology. 22, 245702−245708. Wijaya, E., Lenaerts, C., Maricot, S., Hastanin, J., Habraken, S., Vilcot, J. P., 18
Boukherroub, R., Szunerits, S. 2011. Curr. Opin. Solid State Mater. Sci. 15, 208−224. Xiang, Q., Cheng, B., Yu, J. 2015. Angew. Chem. Int. Ed. 54, 11350−11366. Xu, Y., Li, K., Qin, W., Zhu, B., Zhou, Z., Shi, J., Wang, K., Hu, J., Fan, C., Li, D. 2015. Anal. Chem. 87, 1968−1973. Xue, T.; Cui, X.; Guan, W.; Wang, Q.; Liu, C.; Wang, H.; Qi, K.; Singh, D. J.;Zheng, W. 2014. Biosens. Bioelectron. 58, 374−379. Yan, H., Cheng, H., Yi, H., Lin, Y., Yao, T., Wang, C., Li, J., Wei, S., Lu, J. 2015. J. Am. Chem. Soc. 137, 10484−10487. Yang, H., Lin, C. W., Hua, M. Y., Liao, S. S., Chen, Y. T., Chen, H. C., Weng, W. H., Chuang, C. K., Pang, S. T., Ma. C. C. M. 2014. Adv. Mater. 26, 3662−3666. Yu, X., Li, Y., Wu, J., Ju, H. 2014a. Anal. Chem. 86, 4501−4507. Yu, X., Park, H. S. 2014b. Carbon. 77, 59−65. Yuan, P., Deng, S., Xin, P., Ji, X., Shan, D., Cosnier, S. 2015. Biosens. Bioelectron. 74, 183−189. Zagorodko, O., Spadavecchia, J., Serrano, A. Y., Larroulet, I., Pesquera, A., Zurutuza, A., Boukherroub, R., Szunerits, S. 2014. Anal. Chem. 86, 11211−11216. Zhang, Q., Xu, X., Li, H., Xiong, G., Han Hu, H., Fisher, T. S. 2015. Carbon. 93, 659−670. Zhang, Y., Bai, X., Wang, X., Shiu, K. K., Zhu, Y., Jiang, H. 2014. Anal. Chem. 86, 9459−9465. Zhu, N., Han, S., Gan, S., Ulstrup, J., Chi, Q. 2013. Adv. Funct. Mater. 23, 5297−5306.
19
Figure 1 (A) CV (blue) and ESPR (red) responses during the eletrodeposition of GO, curve a: 1st and b: 500th cycle. (B) SPR plot in consistence with ESPR. (C) SPR signals of dsDNA/NTPy/rGO in a function of CV loops. (D) SEM images of bare Au (a) and rGO/Au (b) chips. Figure 2 (A) SPR baselines of bare Au (a), rGO/Au (b), NTPy/rGO/Au (c), and cDNA/NTPy/rGO/Au (d). (B) Real-time angle shifts of dsDNA/NTPy/rGO/Au (a), dsDNA/MPA/Au (b), bare Au (c), dsDNA/Au (d), and HRP-SA/NTPy/rGO/Au (e). The arrows and texts indicate where and how to proceed the stage of interest. Figure 3 AFM images of (A) rGO/Au, (B) NTPy/rGO/Au, and (C) dsDNA/NTPy/ rGO/Au. Figure 4 (A) Sampling points on real-time trajectory: cDNA/NTPy/rGO/Au (a), dsDNA/NTPy/rGO/Au (black) and cDNA/NTPy/rGO/Au+PBS (red) (b), rDNA/ dsDNA/NTPy/rGO/Au
(c),
HRP-SA/rDNA/
dsDNA/NTPy/rGO/Au
(d),
and
(d)+aniline/H2O2 (e). The arrows and texts indicate where and how to proceed the following stage. (B) SPR responses, (C) CVs and (D) SEM images before (a) and after (b) the polymerization of aniline. Inset B: zoom-in of (b). Figure 5 (A) The calibration curve (concentration of tDNA: 1.0 10.0, 100.0 fM, 1.0, 10.0, 100.0 pM, and 1.0 nM, from bottom to top). (B) Normalized SPR intensities of tDNA and, 1-, 2-, and 3-Base mismatches (0.1 nM). Inset: The corresponding SPR responses. Scheme 1 Schematic illustration of the noncovalent functionalization of rGO for sensitizing SPR-Based DNA sensing synergistically with biocatalytic polymerization.
20
21
Figure 1
22
Figure 2
23
Figure 3
24
Figure 4
25
Figure 5
Highlights
An ultrasensitive SPR-based biosensing mode was developed with dualamplifiers incorporated within both the substrate and the tracing tag, which synergized altogether to accomplish a femtomolar-level DNA detection.
Nickel-chelated nitrilotriacetic pyrenes supported by electro-reduced graphene nanosheets were in situ self-assembled noncovalently on SPR chip for highly ordered and densely populated immobilization of capture probes.
Aniline was selected as a real-time initiator for biocatalytic polymerization by labeled enzymes. Its oxidative product of polyaniline triggered mass effect upon the above interface, jointly resulting in a significant sensitization on SPR output.
26