In this study, an unconventional antenna-like heterostructure comprised of arrays of nanoporous Prussian blue (PB) nanocube heads/TiO2 nanowire (NW) arms (PB-TiO2) is developed for efficient three-dimensional interfacial sensing of small molecules and cellular activities. Inspired by insect tentacles, which are comprised of both target recognition and signal transduction units, one-dimensional TiO2 NW arrays are grown, followed by selective growth of nanoporous PB nanocubes on the tips of the NW arrays. Due to their high selectivity and bioaffinity toward cells, long biostability under a cell culture adhesion condition (up to 108 h) is obtained, and with its inherent bio-mimetic enzymatic activity, the obtained nanoporous PB nanocubes (head segment) serve as robust substrates for site-selective cell adhesion and culture, which allows for sensitive detection of H2O2. Simultaneously, the single-crystalline TiO2 NWs (arm segment) provide efficient charge transport for electrode substrates. Compared with PB-functionalized planar electrochemical interfaces, the PB-TiO2 antenna NW biointerfaces exhibit a substantial enhancement in electrocatalytic activity and sensitivity for H2O2, which includes a low detection limit (∼20 nM), broad detection range (10−8 to 10−5 M), short response time (∼5 s) and long-term biocatalytic activity (up to 6 months). The direct cultivation of HeLa cells is demonstrated on the PB-TiO2 antenna NW arrays, which are capable of sensitive electrochemical recording of cellular activity in real time, where the results suggest the uniqueness of the biomimic PB-TiO2 antenna NWs for efficient cellular interfacing and molecular recognition.
Tentacles, which are the long, functional antennas of insects, provide unique and sensitive signal detection capabilities, such as olfaction, gestation and touching, which can guide the motion and functions of insects.1 The tip of a tentacle, known as the flagellum, is covered with ultra-sensitive olfaction sensing units that can detect specific molecular targets at extremely low concentrations (as low as several molecules per liter). The signal is then transferred through the trunk segment of the tentacle to the neuron networks.2 This dual-functional antenna structure of the insect tentacles demonstrates a unique modality for signal capture and transduction and could inspire new electrochemical sensor designs for enhanced sensitivity and performance.3 In particular, a heterostructure comprised of an ultra-sensitive redox unit and a rapid charge transport unit could substantially expand the capabilities of positioning and targeting of cell patterning and in detecting fluctuations and functions of target molecules at an unprecedented level.
Numerous cellular functions are closely correlated with small molecules, such as O2,4 glucose,5 glutathione,6 NO,7 CO8 and H2O2,9 for enzymatic co-factors, signaling and expression. Among them, H2O2 is an important reactive oxygen species that can diffuse across cell membranes and lead to oxidative protein modification.10 Prussian blue (PB) is known as an artificial enzyme peroxidase with porous frameworks, which can efficiently reduce H2O2 at extremely rapid catalytic rates with low overpotentials.11 For example, molecularly imprinted PB films12 and PB/carbon nanotubes13 have been used for biosensing because their low overpotentials can exclude interferences from coexisting substances, such as ascorbic acid (AA), uric acid and glucose. However, previous work predominantly used PB nanocrystals that had been post-modified on flat substrates, and thus, only a limited number of crystal faces were exposed to electrolytes, which resulted in lower electrochemical responses. In addition, a relatively large background current still existed due to the secondary electrochemical reactions that occurred on the planar electrode surface.14 Although bio-mimetic enzyme-based electrical sensors are attractive tools, their use on a robust lab-on-a-chip platform with fast, sensitive and stable three-dimensional (3D) porous interfaces for localized real-time, long-term monitoring of cellular activities and physicochemical changes have not been demonstrated.15, 16, 17, 18, 19
Inspired by insect tentacles, herein, we developed an unconventional antenna-like heterostructure based on direct growth of porous PB nanocube heads on the tips of TiO2 nanowire (NW) arms, which can be used for 3D interface recognition and biosensing. Single-crystalline TiO2 NWs are first hydrothermally grown on conducting substrates, followed by the direct growth of nanoporous PB nanocubes on the tips of the TiO2 NW arrays via an etching and seed-assisted process (Figure 1, Supplementary Figure S1). The PB nanocubes (head segment) provide site-selective cell adhesion and growth, a large surface area for catalysis and a high sensitivity and selectivity for H2O2. The semiconductor TiO2 NW arms offer fast charge transport from the PB redox centers to the underlying electrode surface due to the excellent electron mobility of TiO2, the continuous one-dimensional charge transport channels20, 21 and the low background electrical current noise. As a proof-of-concept design, the bio-mimetic PB/TiO2 antenna NW heterostructures exhibit high selectivity and bioaffinity toward living cells and excellent biostability under cell culture adhesion conditions (up to 108 h). Compared with that of PB-functionalized planar electrochemical interfaces, the electrocatalytic activity on H2O2 produced by HeLa cells is enhanced by ∼550 times on the PB-TiO2 antenna NWs, which exhibits a high sensitivity of ∼20 nM, broad detection range from 10−8 to 10−5 M and a fast response time that is within 5 s.
Materials and methods
AA, dopamine, uric acid and Phorbol 12-myristate-3-acetate (PMA) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Tetrabutyl titanate, hydrochloric acid (37 wt%), K4Fe(CN)6·3H2O, K3[Fe(CN)6]·3H2O, NaNO2, Na2SO3 and H2O2 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Dulbecco's Modified Eagle's medium (high glucose), RPMI-1640, fetal calf serum, penicillin G, streptomycin and trypsinase were purchased from GIBCO BRL (Grand Island, NY, USA). The fluorine-doped tin oxide-coated glass ∼14 Ω per square substrates was purchased from Wuhan Ge-ao Ltd. (Wuhan, China). Other reagents were of analytical grade and used as purchased. All the solutions were prepared by Milli-Q water (Merck KGaA, Darmstadt, Germany) and deaerated with high-purity nitrogen before experiments.
The PB-TiO2 NWs were prepared by a two-step interfacial growth method. TiO2 NWs were first grown on the fluorine-doped tin oxide-coated glass (∼14 Ω per square substrates, Wuhan Ge-ao Ltd.) by a hydrothermal method, as described previously.22, 23 Then, the TiO2 NWs glass substrates were placed within a 125-ml glass bottle, containing 80 ml of HCL (0.005 M) and 136 mg of K3[Fe(CN)6]·3H2O, and stirred for 30 min. The bottle was placed in an oven at 85 °C for 24 h to form the PB nanocrystal seeds. The TiO2 NWs with PB seeds were washed with neat ethanol and water thoroughly to remove the adsorbed nanocrystals on the TiO2 NWs surface. To form the PB hierarchical structure on TiO2 NWs, K4Fe(CN)6·3H2O (125 mg) was first added to a HCl solution (0.05 M, 80 ml) under stirring for 30 min. Then, the TiO2-PB seeds substrate was slowly immersed into the above mixture. After that, the container was placed into an oven and heated at 85 °C for 6–18 h. The obtained PB-armed TiO2 NWs was taken from the container, washed with deionized water and dried in a vacuum oven at 70 °C for 12 h.
Results and discussion
Fabrication of PB-TiO2 antenna NW heterostructures
The controlled synthesis of the PB-TiO2 antenna NW heterostructures is performed in two steps. One-dimensional TiO2 NWs are first hydrothermally grown on fluorine-doped tin oxide substrates, followed by a seed-induced growth of PB nanocubes on the tips of the pre-formed TiO2 NWs (Experimental section in the Supplementary Information). Scanning electron microscopy images show that the diameters and densities of the TiO2 NW arrays are well tuned by the acidity of the precursor solution.22, 23 Increasing the solution acidity leads to a decrease in both diameter and density (Figure 2a, Supplementary Figures S2a and c). Subsequently, the tips of the TiO2 NWs are etched using diluted HCL, followed by hydrothermal growth of PB nanocubes (Supplementary Figure S1).24 At low TiO2 NW densities, each NW tip is covered with a cluster of nanocubes, which substantially exceed the diameter of the corresponding NW arms (Supplementary Figure S2b). At intermediate NW densities, the measured average diameter and interspacing between adjacent NWs are ∼100 and 150 nm, respectively (Figure 2a). The nanocubes with well-defined shapes are grown from each TiO2 NW (Figure 2b, Supplementary Figure S3). Cross-sectional scanning electron microscopy images also clearly show the bio-mimetic antenna-like heterostructure formation (Figures 2c and d). Compared with bare TiO2 NWs, the NW tips are covered by a thin layer of PB nanocubes. The edge length of the nanocubes is calculated to be ∼150 nm, which is similar to the average interspacing between adjacent TiO2 NWs. The top-view and side-view transmission electron microscopy (TEM) images show that the number of nanocubes and underlying TiO2 NWs are similar, which suggests that essentially, each NW tip serves as a growth base for individual nanocubes (Figures 2e and f). This structure provides sufficient space surrounding the crystallized PB nanocubes while maintaining a close contact between the PB nanocubes with the TiO2 NW bases. When the diameter and density of the TiO2 NWs are further increased, the interspacing between adjacent NWs is less than the average NW diameter. Thus, the nanocubes on the NW tips accumulate, which forms a multi-layered nanocube assembly (Supplementary Figure S2d). The heterostructure of the PB nanocube head/TiO2 NW arm is revealed by TEM (Figure 2g, Supplementary Figure S4). The single TiO2 NWs (Figures 2h and i) and single PB nanocubes (Figures 2j and k) in the heterostructures are further characterized by high-resolution TEM (HRTEM). Both the HRTEM and selected area electron diffraction pattern (Figure 2l) demonstrate the crystallized characteristics of the TiO2 NWs and PB nanocrystals.
The crystal structure of the PB-TiO2 antenna NW heterostructures is further examined by X-ray diffraction, which shows a combination of the diffraction peaks of TiO2 and PB (Figure 3a). No additional peaks other than those of fluorine-doped tin oxide substrates are observed, which indicates the high purity of the samples. Compared with pristine TiO2 NWs that have negligible absorption in the visible range, the ultraviolet–visible spectra of the PB-TiO2 antenna NWs show a substantial absorption increase in the range of 500−900 nm with a maximum at 714 nm, which corresponds to the inter-metal charge transfer bands from Fe2+ to Fe3+ in PB nanocrystals (Figure 3b).25 The corresponding energy dispersive X-ray analysis of the PB-TiO2 NWs shows clear Fe and Ti signals (Supplementary Figure S5). The growth of PB-TiO2 antenna NWs is further demonstrated by the infrared spectra. In addition to the infrared absorption of pristine TiO2 NWs centered at 3428 and 1628 cm−1, a new absorption band at 2095 cm−1 is observed for the PB-TiO2 antenna NWs, which corresponds to the C≡N stretching mode in the FeII(C≡N)/FeIII pair of PB nanocrystals (Figure 3c).26 Furthermore, the Raman spectrum of the PB-TiO2 antenna NWs shows the characteristic band of PB at 2157 cm−1, which further confirms the successful incorporation of PB over TiO2 NWs (Figure 3d).13
The synthesis mechanism of the proposed etching and seed-assisted process is as follows. First, the intermediate PB seed-modified TiO2 NWs are observed on the tips of pristine TiO2 NWs, in which the composite TiO2 NWs have identical close-packed cubic structures (Supplementary Figure S6). Thus, no clear phase separation occurs on the TiO2 NW interfaces during the hydrothermal growth process. The removal of PB nanocrystals by an alkaline solution at room temperature for 5 h from the PB-TiO2 NWs increased the porous property on the top of the TiO2 NWs (Supplementary Figure S7), which suggests the existence of acid etching during the hydrothermal process. For comparison, the growth without PB seeds does not result in any nanocube structures on the tips of the TiO2 NWs (Supplementary Figure S8). However, the growth with acid etching only and in the absence of seeds shows that no nanocube clusters formed on the TiO2 NWs during the hydrothermal process (Supplementary Figure S9); only sporadic PB nanocrystals are adsorbed on the surface of the TiO2 NWs.
The electrochemical properties of the PB-TiO2 antenna NW heterostructure are investigated by the cyclic voltammetry method. For comparison, the pristine TiO2 NWs are also measured under similar conditions. No redox peaks except for the capacitive current are observed for the pristine TiO2 NW electrode, whereas the PB-TiO2 antenna NW electrode has a pair of redox peaks at 0.23 and 0.18 V (Figure 4a), which corresponds to the reversible conversion of PB to Prussian white.25 The scan rate dependency for the voltammetry profile of the PB-TiO2 antenna NW electrode in the range 50−1000 mV s−1 is then presented (Figure 4b), in which the electrochemical stability of the PB-TiO2 antenna NWs is demonstrated by repeating cyclic voltammetry measurements at a scan rate of 50 mV s−1. There is no observable difference in either current level or peak positions for the cyclic voltammetry curves after 50 cycles (Figure 4c), which confirms the stable structure of the immobilized PB nanocubes on the TiO2 NW tips.
The capability of using PB-TiO2 antenna NWs as an amperometric sensing modality for H2O2 is further investigated because it is well-known that the reduced form of PB exhibits a high catalytic activity for H2O2 reduction.27 During this process, PB acts as an electron transport mediator between the electrode and H2O2 in a solution. The presence of H2O2 (5 mM in phosphate buffer saline (PBS), pH of 6.0) leads to a clear increase in current density (corresponding to H2O2 reduction) at a lower overpotential (that is, −50 mV versus Ag/AgCl) (Figure 4d). The calculated electron transfer rate constant, ks, is 5.72±0.05 s−1, which is much greater than that for most of the H2O2 electrodes reported previously, such as the ordered nanoporous niobium oxide film (0.28 s−1),28 NaY zeolite/glassy carbon electrode (0.78±0.04 s−1)29 and colloidal Au/carbon paste (1.21±0.08 s−1).30
PB-TiO2 antenna NW arrays for H2O2 sensing
The sensor performance of PB-TiO2 antenna NWs in detecting H2O2 is extensively investigated. Because coexisting molecular interferences, such as AA, O2 and so on, may affect the electrochemical determination of H2O2, the bias potential should be prudently selected to optimize the cathodic current and sensitivity obtained at the PB-TiO2 antenna NW electrodes. Amperometric experiments were performed to investigate the responses of PB-TiO2 antennas from H2O2 at various potentials of −0.10, −0.05, 0.00, 0.05 and 0.10 V (versus Ag/AgCl). Several interference molecules, which included O2, Na2SO3, uric acid, 3,4-dihydroxyphenylacetic acid, NaNO2 and AA, were tested (Figure 5a). In general, a low anodic current is obtained for the interference molecules at relatively negative potentials. For example, the ratio of anodic current between H2O2 to AA (0.1 mM each) increases from 6.8 to 55 A when the applied potential is reduced from 0.10 to −0.05 V (versus Ag/AgCl), which increases the selectivity.31 Hence, −0.05 V (versus Ag/AgCl) is selected as the optimized operational bias potential. In contrast, control experiments of pristine TiO2 NWs and TiO2 NWs post-modified with PB (TiO2 NWs+PB) do not show a similar high current signal or signal ratios, even at an applied potential of −0.05 V (Figures 5b and c), which suggests that the direct growth and attachment of PB nanocubes on TiO2 NW tips enhance the sensitivity and selectivity. In addition, these data confirm the bio-mimetic enzymatic amplification nature of the H2O2 catalysis at the PB-TiO2 antenna NW electrodes. The long-term stability of PB-TiO2 antenna NW electrodes is exemplified by repeating cyclic voltammetry cycles at different bias voltages. The PB-TiO2 antenna maintains 95% of its initial signal responses, even after 1000 cycles (Figure 5d), which is much more sensitive and stable than pristine TiO2 NWs and TiO2 NWs post-modified with PB, which is excellent for long-term signal monitoring (Figures 5e and f).
The amperometric measurement is further investigated by determining the electrocatalytic activity of the PB-TiO2 antenna NWs for H2O2. A substantial cathodic current change is observed at the PB-TiO2 antenna NW electrodes at −50 mV when 25 μM of H2O2 is introduced in the buffer, whereas essentially no response is obtained at the pristine TiO2 NWs under otherwise similar conditions (Figure 5g). The contribution of the observed anodic current to the catalysis of H2O2 is confirmed by adding catalase,10, 32 a selective scavenger of H2O2, into the solution with H2O2. A large decrease in the cathodic current to essentially the background level is observed, which indicates that the signal response can be ascribed to the catalysis of H2O2 by the PB-TiO2 antenna NW electrodes.
Amperometric responses of the PB-TiO2 antenna NWs with successive addition of H2O2 are conducted at the optimized potential of −50 mV. The stepwise current signal correlates well with each addition of H2O2, whereas an almost negligible current response is observed for the pristine TiO2 NWs (Figure 5h). The calibration plot of the current change obtained at the PB-TiO2 antenna NWs with the H2O2 concentration is summarized (Figure 5i), and the analytical performance of the PB-TiO2 antenna NW-based H2O2 biosensor at the optimum conditions is listed in Supplementary Table S1. Other previously reported H2O2 biosensors based on TiO2 or titanate nanostructures are also listed in Supplementary Table S1 for comparison. The PB-TiO2 antenna NWs exhibit an excellent sensor performance, which includes their higher selectivity, wider linear detection range and lower detection limit. Specifically, the dynamic linear range of 10−8 to 10−5 M to detect H2O2 at the applied potential of −50 mV is significantly wider than those obtained in previous H2O2 biosensing interfaces33, 34 and fulfills the requirement of real-time, long-term tracking of an H2O2 concentration (Figure 5i). Moreover, the PB-TiO2 antenna NW biosensors can be tested and stored at room temperature and can be repeatedly tested for over 180 days without substantial degradation (Supplementary Figure S10), which suggests the high stability of the PB-TiO2 antenna NW electrodes.
Cell culture and extracellular H2O2 sensing
The capability of PB-TiO2 antenna NW electrodes for direct growth of cells and extracellular H2O2 detection is further demonstrated. The uniform coverage of PB nanocubes offers an excellent substrate for cell attachment and growth, which can subsequently produce H2O2 via different cell functions (Figure 6a).35, 36, 37 The fluorescence images of HeLa cells grown on top of the PB-TiO2 antenna NWs clearly confirm a good coverage of cells on the NWs (Figure 6b). The cell viability is measured by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) in a Nun Immune Omni Tray (Angle Nun International, Rochester, NY, USA; Experimental Section in Supporting Information, Supplementary Figures S11–S13), which shows that a high HeLa cell viability (>80%) is maintained for growth for up to 5 h (Figure 6c). The measured amperometric responses obtained at the bio-interface between the PB-TiO2 antenna NWs and HeLa cells are 50 mM PBS (pH of 6.0) at an applied potential of −50 mV versus Ag|AgCl (Figure 6c). When 50 mM of PMA is injected into the HeLa cell-NW assay, an increase in the cathodic current is observed (Figure 6d). This phenomenon is attributed to the effect of PMA for inducing H2O2 production from the cells.10 An anodic current increase of ∼32.5 μA is obtained in 15 s, which is similar to the time scale reported previously.38 No response is observed at the bare TiO2 NWs with the same addition of PMA. In addition, the injection of the catalase solution (300 U ml−1 in PBS) reduces the current level to almost the background current level; catalase is known to inhibit the PMA function.10 Accordingly, the increase in cathodic current at the PB-TiO2 antenna NW bio-interface located near the cells is ascribed to the enzymatic reduction of H2O2, which is effectively mediated by the PB nanocubes grown on the TiO2 NWs interface.
The much enhanced sensitivity of the bio-mimetic PB-TiO2 antenna NWs is attributed to the unique bio-inspired head/arm heterostructure, in which intimate contact between the PB nanocube heads and the TiO2 NW arms can create synergistic properties of both components. The porous PB nanocubes offer a robust substrate for site-selective cell adhesion and cultivation of living cells because the porous nanocubes exhibit high selectivity and bioaffinity toward cells and have excellent biostability under the cell culture adhesion condition (up to 108 h) (Supplementary Figure S14). Furthermore, the porous nanocrystals can also serve as long-term stable and sensitive sensing elements for H2O2 due to their inherent bio-mimetic enzymatic activity, high surface area and 3D stereo space-based signal molecules for touching and recognition. In addition, compared with conventional planar PB-covered electrochemical interfaces,39, 40 the electrocatalytic activity is enhanced at the PB-TiO2 antenna NW biointerfaces due to the rapid charge transport realized by the one-dimensional TiO2 NW structure. Thus, this PB-TiO2 antenna NW demonstrates a new sensing platform to reliably and durably detect extracellular molecules.
In summary, a bio-mimetic PB-TiO2 antenna NW array-based bio-interface is demonstrated for the first time as an excellent structure that can recognize 3D signal molecules and that can be used for biosensing. Living cell adhesion and in-situ cultivation on PB-TiO2 antennas, which integrates with sensitive real-time monitoring of cellular messenger molecules, are realized by the dual-functional PB nanocube heads and TiO2 NW arms. The optimized PB-TiO2 antenna NW biointerfaces exhibit a remarkable sensing performance in detecting H2O2 with a high sensitivity and selectivity, a broad detection range from 10−8 to 10−5 M, a low detection limit down to 20 nM and a short response time that is within 5 s. In addition, bio-mimetic PB nanocrystals are stably anchored on the TiO2 NWs and can maintain long-term biocatalytic activity (up to ∼180 days). The capability of the PB-TiO2 antenna NW biointerfaces in determining extracellular H2O2 released from human tumor cells is further demonstrated. This work not only provides a method for 3D interface recognition and biosensing on the hierarchical bio-mimetic nanostructured semiconductors but also suggests a general approach for durable, reliable biomolecule detection in biological systems.
We thank the following funding agencies for supporting this work: the National Key Basic Research Program of China (2013CB934104, 2012CB224805), the NSF of China (20890123, 21322311, and 21071033), the Program for New Century Excellent Talents in University (NCET-10-0357), the Program for Professor of Special Appointment (Eastern Scholar) at the Shanghai Institutions of Higher Learning and the Deanship of Scientific Research of the King Saud University (IHCRG#14-102) and the Shanghai Science and Technology Municipality (14JC1490500). BK and JT thank the Scholarship Award for Excellent Doctoral Student granted by the Ministry of Education of China and the Interdisciplinary Outstanding Doctoral Research Funding of the Fudan University.
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Supplementary Information accompanies the paper on the website (http://www.nature.com/am)
Journal of Materials Chemistry B (2018)