Smart polymer-based calcium-ion self-regulated nanochannels by mimicking the biological Ca2+-induced Ca2+ release process

In nature, ion channels play key roles in controlling ion transport between cells and their surroundings. Calcium ion (Ca2+)-induced Ca2+ release (CICR), a critical control mechanism for Ca2+ channels, occurs due to a Ca2+ concentration gradient working in synergy with ryanodine receptors, which are famously known as “calcium sparks”. Inspired by this self-regulated biological process, a smart Ca2+ concentration-modulated nanochannel system was developed by integrating a poly{N-isopropylacrylamide-co-acrylamide-[4-(trifluoromethyl) phenyl]-2-thiourea0.2-co-acrylamide-DDDEEKC0.2} (denoted as PNI-co-CF3-PT0.2-co-DDDEEKC0.2) three-component copolymer onto the nanochannels of a porous anodic alumina (PAA) membrane. In this smart polymer design, the DDDEEKC hepta-peptide unit has an extraordinary binding affinity with Ca2+ through coordination bonds, while CF3-PT functions as a hydrogen bond mediation unit, facilitating the remarkable conformational transition of the PNI main chain in response to Ca2+-specific adsorption. Due to these futures, the dynamic gating behaviors of the modified nanochannels could be precisely manipulated by the Ca2+ concentration. In addition, the sensitive Ca2+ response, as low as 10 pM with a high specificity toward Ca2+ capable of discriminating Ca2+ from other potential interference metal ions (e.g., K+, Cu2+, Mg2+, Zn2+, Fe3+, and Al3+), remarkable morphological change in the nanochannel and satisfactory reversibility indicate the great potential of Ca2+-responsive polymers for the fabrication of biodevices and artificial nanochannels. A calcium-responsive polymer that mimics the function of pore-forming membrane proteins found in muscle cells offers a new platform for biotechnology and regenerative medicine. Inspired by the types of calcium channels that regulate muscle contraction, a team led by Guangyan Qing from the Dalian Institute of Chemical Physics, China, created a three-component smart material. It includes a seven amino acid peptide that recognizes calcium ions, a chemical that mediates conformational changes, and a flexible polymer that opens and closes the nanochannel in response to changes in calcium levels. The researchers showed that the material changed shape controllably and reversibly in response to minute changes in calcium concentrations. They propose the smart polymer could have many applications, including, for example, microfluidics, biosensors and bioseparation devices. Inspired from Ca2+-induced Ca2+ release process responsible for Ca2+ channel and muscle contraction, a smart Ca2+ self-regulated nanochannel system is constructed by the convergence of a Ca2+-responsive polymer with porous anodic alumina membrane. Dynamic gating behavior of the nanochannel could be precisely modulated by Ca2+ concentration utilizing remarkable conformational transition of the copolymer chains from globule to coil.


Introduction
Natural ion channels, with high fidelity to specific ions, act as a pivotal part in supporting the metabolism of living cells (Scheme 1), and their dysfunction can lead to various diseases 1,2 ; 40% of clinical drugs are designed to target ion channel proteins. Among ion channels, Ca 2+ channels are prerequisites for an extensive range of life processes 3,4 , such as functions of nervous systems, cell proliferation, genetic transcription, and muscular contraction 5,6 . In particular, ryanodine receptors (RyRs) on sarcoplasmic reticulum (SR) membranes, which are in charge of the release of intracellular Ca 2+ stores, are sensitive to the concentration of Ca 2+ (Scheme 1, the lower part). Once the concentration reaches a threshold (10 −6 -10 −5 mol L −1 (M)), conformational switching of RyRs will occur, which increases the helicity of the protein and shortens the distance and dihedral angle between two S6-peptide chains (Scheme 2a) [7][8][9] , resulting in the opening of nanopores of RyRs and the release of a large number of Ca 2+ ions from the SR. These released Ca 2+ will bind with calmodulin protein, subsequently leading to the closure of the Ca 2+ channel. Thus, a Ca 2+ -induced Ca 2+ release (CICR) process proceeds, which is crucial to the maintenance of the intracellular Ca 2+ concentration and controls muscle contraction, particularly the cardiac muscle. The malfunction of the CICR process can lead to a great number of serious diseases 10,11 . This unique biological event has inspired chemists to develop various Ca 2+ -sensitive ion channels and nanodevices [12][13][14] .
In the past decade, several elegant approaches have been applied to construct Ca 2+ -modulated nanochannels 15-17 . As typical examples, Siwy et al. 18 reported a Ca 2+ -induced voltage gating, ionic current oscillations 19 , and charge inversion 20 , on the basis of track-etched asymmetric nanopores on polyethylene terephthalate (PET) films. Meng et al. 21 utilized calcein-modified PET nanochannels to achieve a Ca 2+ -responsive nanogating. These modulation strategies mainly rely on surface charge changes of the nanochannels to achieve "on-off" switching, which are different from the ion channels in vivo whose gating behaviors are mainly achieved by the conformational transition of channel proteins, as illustrated in Scheme 2a. Within this context, much attention has been devoted to smart polymer-based 22 nanochannels, which bear the obvious advantages of high controllability, satisfactory reversibility, and remarkable conformational transition in response to external stimuli 23,24 . By producing diverse external stimuli, such as temperature, pH, light, or redox potential [25][26][27] , reversible tuning of ion transport across smart polymer-modified nanochannels can be achieved, which has resulted in wide applications for both material sciences and life sciences [28][29][30][31][32] .
Nevertheless, to the best of our knowledge, smart polymer-based nanochannels that can be manipulated by Ca 2+ concentration have rarely been reported. Building Scheme 1 Illustration of the types of calcium-ion (Ca 2+ ) channels in cells, including voltage-gated calcium channels, ligand-gated calcium channels (e.g., cAMP-modulated Ca 2+ channels), inositol-1,4,5triphosphate receptor (IP 3 R)-modulated Ca 2+ channels, and RyRmodulated Ca 2+ channels (CICR process). CICR is typically a positivefeedback system. First, voltage-gated Ca 2+ channels are activated to allow a small amount of Ca 2+ to pass through the cell membrane. Ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) are sensitive to the above Ca 2+ influx and open the nanochannels, which cause a large amount of Ca 2+ stored in the SR to be released into the cytosol. A remarkable increase in cytosolic Ca 2+ activates muscle contraction. a Structural transition of the S6 linker of the RyR1 protein activated by the binding of Ca 2+ . The outward movements of the U-motif and Cterminal domains (CTDs) trigger an outward movement of the inner S6 helices, presenting as an increase in the dihedral angle (θ). Thus, the hydrogen bond (E4948-Q4949), which helps to keep the closed state of the ionic channel, will be broken, and the nanochannel is opened. b, c Schematic illustration of Ca 2+ -adsorption-induced globule-to-coil transition of the copolymer chains (b) determines the open and closed state of the ionic channel (c). Strong coordination binding between hepta-peptide DDDEEKC and Ca 2+ destroys the initial hydrogen bond network that is constructed by the heptapeptide and the neighboring [4-(trifluoromethyl) phenyl]-2-thiourea (CF 3 -PT), which triggers the conformational transition of the copolymer chains. The expansion of the copolymer chains will block the nanochannel and substantially decrease the ionic flux biomimetic Ca 2+ -sensitive ion channels is important for simulating the self-regulating and gating behaviors of RyR channels, the largest known calcium ion channel in humans. These channels will contribute to a more comprehensive understanding of Ca 2+ signal pathways and promote numerous interesting applications in tissue engineering, controllable drug release, bioseparation, biosensors, microfluidics, and microreactors 33 . Herein, inspired by the CICR process, a biomimetic Ca 2+ self-regulated nanochannel system is constructed based on a Ca 2+ -responsive polymer design. A threecomponent copolymer PNI-co-CF 3 -PT 0.2 -co-DDDEEKC 0.2 was designed according to a "recognition-mediation-main chain" concept 34,35 , in which the hepta-peptide DDDEEKC works as a Ca 2+ -recognition unit, CF 3 -PT functions as a hydrogen-bond mediation unit and PNI provides a flexible polymer main chain. As the core recognition unit for Ca 2+ , DDDEEKC with an α-helix conformation was first found as an N-terminal region of statherin 36 with a strong adsorption capacity toward hydroxyapatite, which inspired us to utilize this natural peptide to design the Ca 2+ -binding material 37,38 . Subsequently, CF 3 -PT was introduced as a mediation unit owing to its strong hydrogen bond donating capacity. The thiourea group in CF 3 -PT can combine with the carboxyl groups in the above hepta-peptide utilizing multiple hydrogen bonding interactions to build a compact hydrogen bond network within the copolymer, as shown in Scheme 2b in the left panel. When the copolymer film is exposed to Ca 2+ , multiple and strong chelation bindings between Ca 2+ and the carboxyl groups in the hepta-peptide gradually destroy the initial hydrogen bond network. When the Ca 2+ concentration reaches a threshold, the copolymer chains undergo a dramatic globule-to-coil transition, leading to an obvious expansion of the copolymer film (Scheme 2b, right panel). This smart polymer design not only improves the grafting density of the core hepta-peptide to provide more binding sites for Ca 2+ but also supplies an ideal platform to amplify the Ca 2+ recognition signal by taking advantage of the conformational transition of the copolymer chain. The combination of these two merits can substantially improve the sensitivity of the material.
Then, porous anodic alumina (PAA) was chosen as a substrate to build the nanochannel system. Compared with other porous materials, PAA exhibits remarkable advantages, such as adjustable film thickness, high porosity, and tunable parameters, and has been widely used in controlling ion transportation, as well as constructing various functional devices [39][40][41] . By using surface-initiated atom transfer radical polymerization (SI-ATRP) 42,43 , the designed copolymer was then immobilized onto the straight-through nanochannel of a PAA membrane with an average diameter ranging from 70 to 100 nm. The affinity test, quartz crystal microbalance with dissipation (QCM-D) binding analysis, electrochemical impedance spectroscopy (EIS) analysis, morphological observations, and transmembrane ionic current measurements clearly demonstrated that the well-developed nanochannel displayed controllable and reversible gating abilities toward Ca 2+ adsorption or desorption, as illustrated in Scheme 2c. It is worth noting that such a gating effect was highly specific to Ca 2+ , and other metal ions, such as K + , Mg 2+ , Al 3+ , Zn 2+ , Fe 3+ , and Cu 2+ , did not induce any evidential changes. This work provides a smart polymer-based design concept to mimic the crucial CICR process; precise Ca 2+ -modulated gating performance might facilitate the development of various bioseparation membranes, nanochannels, and microfluidic devices 44 .

Synthesis of acrylamide hepta-peptide
The synthesis of acrylamide hepta-peptide was illustrated in Scheme 3a. DDDEEK(Dde)C (0.204 g, 0.2 mmol) and 0.2 mL triethylamine were dissolved in 15 mL anhydrous N,N′-dimethylformamide (DMF), and the mixture was stirred for 15 min in an ice bath. Then, 5 mL of DMF containing acryloyl chloride (0.035 mL, 0.4 mmol) was added to the mixture dropwise and stirred for 0.5 h in an ice bath. The reaction mixture was then stirred at room temperature for 24 h. Most DMF was evaporated under reduced pressure. The crude product was purified on a Shimadzu UFLC 20A purity system with a C18 reverse-phase semipreparative chromatographic column. The pure product was obtained as a white powder (0.142 g, yield: 66%). 1  For the modification of the PAA membrane (Scheme 3b), the PAA membrane was first immersed in distilled water and ethanol for 10 min, followed by a quick dip in a hydrochloric acid aqueous solution (5%, v/v) for 35 s and subsequently in hydrogen peroxide heated at 100°C for 1 h to generate surface hydroxyl groups. After that, the membrane was washed with an excess of distilled water and ethanol and dried under a nitrogen flow. Then, it was heated at 65°C in 40 mL toluene containing 1.0 mL APTES for 3 h to obtain chemically bonded-NH 2 groups on the membranes. The reaction was performed in a nitrogen atmosphere. Ethanol was used to wash out the remaining APTES. After drying under a flow of nitrogen gas, the PAA membrane was immersed in 50 mL dry dichloromethane containing 0.4 mL pyridine. Then, 2bromoisobutyryl bromide (0.4 mL) was added dropwise into the solvent at 0°C for 1 h, and then at 25°C for 12 h. After rinsing with CH 2 Cl 2 , a bromine-modified PAA membrane was received.
The modifications of PNI-co-CF 3 -PT 0.2 -co-DDDEEKC 0.2 were synthesized by the method of ATRP according to the literature 43  The reaction was carried out at 60°C for 15 h with nitrogen protection. Subsequently, the copolymermodified PAA membrane was sequentially cleaned with DMF, water, and ethanol and subsequently dried under a nitrogen flow. The PNIPAAm-modified and CF 3 -PT-co-DDDEEKC-modified PAA membranes were prepared through a method similar to that described above. The PNI-co-CF 3 -PT 0.2 -co-DDDEEKC 0.2 -modified Au electrode was prepared using the same method described above. The working electrode, which was a Au electrode modified with PNI-co-CF 3 -PT 0.2 -co-DDDEEKC 0.2 , a Ag/AgCl reference electrode and a graphite auxiliary electrode made up the threeelectrode system. Temperature: 20°C.

Ionic current measurement
A piece of PAA membrane (bare or modified) was mounted between a two-compartment electrochemical cell according to the literature 45 . Ag/AgCl electrodes were used to apply a transmembrane potential across the membrane. The transmembrane ionic current was measured with a Keithley 6487 picoammeter/voltage source (Keithley Instruments) through Ag/AgCl electrodes. The effective area for the ionic conduction measurements was 20 mm 2 . The electrolyte was 0.01 M sodium chloride (NaCl) solution.

Results
First, the binding affinity of the hepta-peptide with CaCl 2 was evaluated by isothermal titration calorimetry (ITC), which has been widely used as a label-free and quantitative technique to detect the thermodynamic parameters of interactions between a small molecule and a biomacromolecule in solution 46 . A classical isothermal calorimetric titration profile of 20 mM Ca 2+ with 1 mM hepta-peptide in pure water at 25°C is shown in Fig. 1a. Strong heat release was observed, corresponding to añ 5:1 binding stoichiometry between Ca 2+ and heptapeptide with stepwise binding constants of K a1 , 773; K a2 , 1030; K a3 , 763; K a4 , 1050; and K a5 , 700 L mol −1 (Fig. 1b), as well as a accumulative K a (K a1 × K a2 × K a3 × K a4 × K a5 ) of 4.46 × 10 14 L mol −1 , which suggested strong and specific complexation. Carbon nuclear magnetic resonance ( 13 C NMR) titration experiments validated this complexation. As shown in Fig. 1c-f, upon the addition of different amounts of CaCl 2 to the hepta-peptide in deuterated water, six sets of carbon atom signals for carboxyl groups in the hepta-peptide display clear downfield shifts. By comparison, no obvious chemical shift change was observed for the carbon atom signals of the amide groups. This difference indicated that multiple coordination bonds between Ca 2+ and the carboxyl groups in the hepta-peptide were the main driving forces for the strong complexation.
A possible binding model was provided by molecular dynamic calculations by using the density function theory. Figure 2a, b shows three-dimensional structures of the hepta-peptide before and after interacting with five Ca 2+ , and some remarkable changes in peptide conformation and dihedral angle (θ) of the carboxyl group could be observed. Specifically, the distance between the amine in the side chain of the sixth Lys and the thiol in the seven Cys decreases from 9.98 to 9.59 Å, while the distance between the carboxyl groups in the fourth Glu and the fifth Glu decreases from 10.97 to 10.61 Å. The dihedral angle of the carboxyl group in the fifth Glu displays a noteworthy increase from −31°to −39°after binding with Ca 2+ . Circular dichroism (CD) was applied to further discuss the conformation change of the hepta-peptide. The CD spectrum of the hepta-peptide was similar to that observed for α-helical polypeptides according to the literature 47,48 . Although the banding intensity may vary with solvents due to the different polarity and hydrogen bonding strength, a strong negative adsorption peak centered at 200 nm and a weak shoulder peak at~225 nm could be observed, which represent the exciton split of the π-π* and n-π at the long wavelength component, respectively (Fig. 2c). Upon the addition of CaCl 2 to the hepta-peptide solution, the CD intensity of the peak at 200 nm decreases slightly, while the signal at 188 nm increases, which indicated a decrease in the proportion of the α-helix structure and the appearance of a random coil structure due to competitive binding interactions between hepta-peptide and the CaCl 2 solute. The remarkable conformational change (Fig. 2d) and high affinity measured by the ITC and 13 C NMR titration experiments revealed that this hepta-peptide can serve as an ideal binding unit for Ca 2+ .
For the purpose of improving the binding affinity with Ca 2+ and amplifying the recognition signal, the acrylamide hepta-peptide was prepared to copolymerize with acrylamide CF 3 -PT and NIPAAm through SI-ATRP, generating a smart PNI-co-CF 3 -PT 0.2 -co-DDDEEKC 0.2 copolymer film (average thickness of 20 nm) on a QCM resonator gold electrode surface. The grafting ratios of 0.2 for hepta-peptide and CF 3 -PT units were determined according to the elemental analysis and integration ratios of the characteristic peaks in the 1 H NMR spectrum of the copolymer ( Supplementary Fig. 2a in SI). To test the three-component copolymer design (Scheme 2b), a fluorescence titration experiment was performed to evaluate the binding affinity (K a ) 49 between a fluoresceinlabeled CF 3 -PT ( Supplementary Fig. 1 in SI) and the hepta-peptide. As shown in Fig. 2e, when 35 equivalents of hepta-peptide were added into the fluorescein-labeled CF 3 -PT (5 × 10 −6 mol L −1 ) in Tris-HCl buffer solution (pH 7.4, 10 mM), and the fluorescence intensity of the solution decreased by 14%, corresponding to a K a of 7969 ± 480 L mol −1 calculated from a nonlinear fitting. It is worth noting that such a fluorescence intensity decrease was not caused by the acidity of the carboxyl groups in the hepta-peptide because the solution pH was maintained at a constant value by a buffer solution. Then, an equimolar mixture of the hepta-peptide and the fluorescein-labeled CF 3 -PT in Tris-HCl buffer solution was prepared, and the fluorescence spectra were recorded (Fig. 2f). Interestingly, when different amounts of CaCl 2 capable of binding with the hepta-peptide were added to the mixture, the fluorescence intensity of CF 3 -PT increased gradually and returned to its initial value prior to the heptapeptide addition. The recovery of fluorescence intensity could be reasonably attributed to the competitive binding of the hepta-peptide with Ca 2+ , which was substantially stronger than that of the hepta-peptide with CF 3 -PT 50,51 . Attenuated total reflection flourier transform infrared spectroscopy (ATR-IR) further validated the interaction between the hepta-peptide and CF 3 -PT ( Supplementary  Fig. 3 in SI).
The above control experiment demonstrated the feasibility of our smart polymer design. Then, the adsorption dynamics of various metal ions on the PNI-co-CF 3 -PT 0.2co-DDDEEKC 0.2 copolymer surface were monitored using a QCM-D to measure the frequency (Δƒ) and energy dissipation (ΔD) 52 . As shown in Fig. 3a by the red line, CaCl 2 (10 μM in pure water) displays slow and strong adsorption on the copolymer surface and reaches a maximum after 45 min (Δƒ = 32.5 Hz); according to the Sauerbrey equation 53 , the adsorption quantity is 191.5 ng cm −2 . Under the same conditions, the FeCl 3 -induced frequency change was only −3.8 Hz, and NaCl, KCl, or AlCl 3 -induced frequency changes were negligible. Interestingly, the adsorption of MgCl 2 and ZnCl 2 was quite similar to that of CaCl 2 in electric charge, leading to abnormal weight loss of the copolymer film, and the QCM frequency slightly increased to 3 Hz. This high Ca 2+ -ion selectivity was further validated by a time-dependent dissipation curve, and the real-time information of the changes in viscoelasticity and thickness of the copolymer layer was recorded. As shown in Fig. 3b (red line), a prominent dissipation increase (ΔD: 5.6 × 10 −6 ) was only observed for CaCl 2 adsorption on the copolymer film. Based on the QCM adsorption theory 54 , a softer and swollen copolymer film after interacting with CaCl 2 was demonstrated by these data, reflecting a relaxed state of the copolymer chains. By comparison, the NaCl, KCl, ZnCl 2 , or AlCl 3 adsorption-induced dissipation changes f Fluorescence spectra of a mixture of fluorescein-labeled CF 3 -PT (5 × 10 −6 mol L −1 ) and equimolar amount of hepta-peptide upon the addition of CaCl 2 in a Tris-HCl buffer solution at 20°C. Recovery of fluorescence intensity indicated that the addition of Ca 2+ ions destroyed the complexation between CF 3 -PT and the hepta-peptide copolymer were negligible. MgCl 2 or FeCl 3 adsorption led to a continuous decrease in the dissipation value (ΔD: −3.6 × 10 −6 or −6.1 × 10 −6 ), revealing remarkable shrinkage of the copolymer film. Distinct adsorption dynamics of various metal ions on the copolymer surfaces and the corresponding dissipation curve variations demonstrated the excellent Ca 2+ selectivity of the copolymer film. The 1 H NMR titration experiment provided auxiliary evidence for complexation between the copolymer and Ca 2+ , in which clear chemical shift changes of the carboxylic acids and amides of the copolymer were observed after the addition of CaCl 2 ( Supplementary Fig. 2b in SI).
The shrinking-to-swelling transition of the copolymer brushes might strongly influence the electrochemical process on the surface of a copolymer-modified gold electrode, resulting in remarkable alteration of the mass  Fig.  3d) 35 . Based on this presumption, the Ca 2+ -adsorptioninduced conformational transition could be monitored by EIS. Figure 3c displays the Nyquist plots obtained for the copolymer-modified electrode before and after being treated with different amounts of CaCl 2 in 5 mM K 3 Fe (CN) 6 /K 4 Fe(CN) 6 solution for 5 min. Initially, the electrochemical activity was blocked by the copolymer on the electrode with a charge transfer resistance (R ct ) of 160 Ω, and this value gradually dropped down to 75 Ω when 100 μM of CaCl 2 was added. This R ct decrease easily avoided the potential interference of nonspecific adsorption, which often results in the increase of R ct . As shown in Fig. 3d, the R ct value decreased gradually with the increasing CaCl 2 concentration, and a 53% R ct decrease was measured upon the addition of 100 μM of CaCl 2 . Notably, even when the concentration of CaCl 2 was only 100 pM, the decrease in R ct (5%) was still obvious and readily detected. In addition, the ion selectivity of the copolymer film was evaluated by EIS measurements, as shown in Fig. 3e, f. The most obvious changes in the Nyquist plot and corresponding R ct were observed when CaCl 2 (100 μM) was tested, while the NaCl, ZnCl 2 , MgCl 2 , CuCl 2 , or AlCl 3 -induced R ct changes were substantially weaker under the same conditions. These data further illustrated the highly selective response of the copolymer toward CaCl 2 .
To construct biomimetic ionic nanochannels, PNI-co-CF 3 -PT 0.2 -co-DDDEEKC 0.2 was grafted onto the porous channel of PAA membranes with uniform straight nanochannels (average pore size: 90-110 nm). The ATR-IR and thermal gravimetric analysis tests validated the modification of the copolymer on the PAA membrane (Supplementary Figs. 4 and 5 in SI). Then, atomic force microscopy (AFM) and scanning electron microscopy (SEM) were adopted to observe the morphological changes of the copolymer-modified PAA membranes after being immersed in a CaCl 2 aqueous solution (20 μM) for 10 min. From the top view of the membrane, as observed by AFM (Fig. 4a, b), the initially well-defined nanopores became indistinct, and a few nanopores were blocked by the expanded copolymers according to the section profiles of the AFM images. According to the statistical analysis of the AFM images, the average pore size decreased from 82.5 to 52 nm, while the root mean square roughness (R q ) of the PAA membrane increased from 7.23 to 10.85 nm, which indicated conspicuous macroscopic surface changes in the copolymer-modified PAA caused by Ca 2+ adsorption. In addition, based on the SEM cross section images (Fig. 4c, d), the average wall thickness of the nanochannels increased from 55 to 81 nm, resulting in a remarkable decrease in the average size of the nanochannels from 67 to 40 nm. These obvious morphological changes provided direct evidence for the expansion of the copolymer chains.
To analyze the elementary composition of the copolymer film, X-ray photoelectron spectroscopy (XPS) was performed. Compared with the XPS spectrum of the bare PAA membrane, signals of C 1s, N 1s, S 2p, and F 1s with binding energies of 285.0, 399.9, 168.4, and 689.0 eV, respectively, could be clearly observed for the copolymermodified PAA membrane, which validated the successful immobilization of the copolymer on the PAA membrane. When the copolymer-modified membrane was treated with CaCl 2 solution (20 μM) for 10 min and then rinsed with water, Ca 2p signals with binding energies of 347.3 and 350.8 eV appeared (Fig. 4e), which indicated that Ca 2+ had been chemically adsorbed onto the copolymer film. From the perspective of C 1s signals, Ca 2+ -adsorption remarkably strengthened the carbonyl signal of C 1s, as shown by the blue dashed lines in Fig. 4f, g, which is a contribution from chelation binding. In addition, the C-H, C-C and C-N signals of C 1s increased substantially, indicating that these functional groups were fully exposed due to the expansion of the copolymer chains 55 .
With the expansion of the copolymer chains, the size of the nanochannels decreased sharply, which further led to a decrease in the ionic flux of the nanochannels. This ionic transport property of the nanochannel was examined by current-voltage measurements using a Keithley 6487 picoammeter (Fig. 5a). All PAA membranes were separately mounted in an electrochemical cell (inset of Fig. 5a). A NaCl solution served as the electrolyte to measure the ionic current across the nanochannels at a constant volume of 1 mL 56 . The initial transmembrane ionic current was 100 μA (at +0.2 V), indicating the good permeability of the copolymer-modified PAA membrane for ion transport. Then, electrolytes with different concentrations of CaCl 2 were separately added to the cell exposed to the PAA membrane. Figure 5b displays the CaCl 2 concentration dependence of the ionic current (at +0.2 V) change ratio [defined as (I − I 0 )/I 0 , where I 0 is the initial current] for the bare, PNIPAAm and PNI-co-CF 3 -PT 0.2 -co-DDDEEKC 0.2 -modified PAA membranes 45 . The detection range for Ca 2+ extended from 1 × 10 −4 mol L −1 to an ultratrace level of 1 × 10 −11 mol L −1 , which fully covered the intracellular and extracellular Ca 2+ / 1 × 10 −8 to 1 × 10 −3 mol L −157,58 and makes it possible to further utilize this artificial nanochannel for medical treatment. For the copolymer-modified PAA membrane, an elegant negative correlation curve between (I − I 0 )/I 0 and the Ca 2 + concentration was observed, suggesting the powerful and flexible regulatory capacity of Ca 2+ for this nanochannel. The ionic current change ratio gradually increased from 12.3% at 10 −11 mol L −1 to 47.5% at 10 −4 mol L −1 , reflecting a satisfactory gating performance. In contrast, a negligible change in the ionic current was observed for the bare or PNIPAAm-modified PAA, which indicated the strong Ca 2+ binding capacity and high grafting density of the hepta-peptide in the threecomponent copolymer contributed to the remarkable sensibility and excellent gating efficiency.
A control experiment was performed to determine the rationality of the three-component copolymer design.
Using hepta-peptide single monolayer and poly(CF 3 -PTco-DDDEEKC 0.5 )-modified PAA membranes, the maximal ionic current change ratios caused by Ca 2+ were only 13.4% and 20%, respectively (Fig. 5c), both of which are substantially lower than that of PNI-co-CF 3 -PT 0.2 -co-DDDEEKC 0.2 -modified PAA. We presumed that Ca 2+ adsorption on the hepta-peptide monolayer surface only changed the surface charge of the nanochannels owing to  20°C (b, d); green lines in the corresponding AFM section profiles. Narrow scan X-ray photoelectron spectra of the copolymer-modified PAA membrane before (f) and after (e, g) treatment with CaCl 2 solution (20 μM) for 10 min at 20°C. e Ca 2p; f, g C 1s. These surfaces were rinsed with water before measurements to eliminate the physical adsorption of Ca 2+ the chelation binding between Ca 2+ and the carboxylic acid groups in the hepta-peptide (Fig. 5d, left panel). For the poly(CF 3 -PT-co-DDDEEKC 0.5 ) two-component copolymer with the absence of PNIPAAm, the flexibility of the copolymer chains was substantially reduced, resulting in a more contracted state owing to strong hydrogen bonding interactions between CF 3 -PT and the hepta-peptide units. Under this condition, the introduction of Ca 2+ could not destroy such a compact hydrogen bond network, and the conformational change was limited ( Fig. 5d, middle panel). Only the integration of heptapeptide, CF 3 -PT, and flexible PNIPAAm into one system allowed the Ca 2+ -triggered globule-to-coil transition of the copolymer to occur and contributed to the remarkable ionic current change (Fig. 5d, right panel).
Satisfactory reversibility of the ionic gating behaviors was also displayed. As shown in Fig. 5e, the ionic current switches between 100 and 50 μA through alternate treatment by the electrolyte with or without CaCl 2 (10 μM), and the reversibility was well maintained after seven cycles. Due to the complexity of the cellular environment, a high demand is set not only for sensitivity and reversibility but also for selectivity of natural ion channels. Thus, the selectivity of an artificial nanochannel system is a vital evaluation criterion. A series of selectivity tests was performed using a control variable method. As expected, not only a sensitive and reversible response to the target Ca 2+ was achieved; this nanochannel system also displayed accurate discrimination capacity among Ca 2+ , K + , Zn 2+ , Mg 2+ , Fe 3+ , Al 3+ , and Cu 2+ (Fig. 5f). The Ca 2+ adsorption-induced ionic current change ratio (47.5%) was significantly larger than that of K + (12.4%) or Zn 2+ (12%) and was distinct from that of Mg 2+ , Fe 3+ , Al 3+ , and Cu 2+ , the adsorption of which led to an increase in the ionic current. This indicates the remarkable advantage of our material with a high specificity toward Ca 2+ that conventional artificial nanochannels have difficulty achieving.

Discussion
In conclusion, inspired by the CICR process, we constructed a smart (PNI-co-CF 3 -PT 0.2 -co-DDDEEKC 0.2 )based calcium-actuated nanochannel. With the intense conformational transitions of the copolymer chains from globule to coil, the gating behavior of the smart nanochannel is closer to that of an ion channel protein, facilitating sensitive monitoring of Ca 2+ concentrations as low as 10 pM and achieving a wide stimulus response range to Ca 2+ that fully covers Ca 2+ levels in human cells. Excellent selectivity among various multivalent metal ions and satisfactory reversibility are attractive features, making it possible to recognize Ca 2+ and modulate the gating of nanochannels in a complex environment. Moreover, this work demonstrates the feasibility of utilizing a biomimetic strategy for building artificial nanochannels with the help of biomolecule-responsive polymer design, which will give rise to more attractive work on biomimetic nanochannels.
In addition to Ca 2+ channels, as one of the most widespread secondary messengers used in signal transduction, Ca 2+ ions play key roles in the physiology and biochemistry of organisms and cells 2 . Particularly, the calcium levels in mammals are tightly regulated, and channels determine the release of Ca 2+ from bone into bloodstream, reabsorption of Ca 2+ in the kidney back into circulation, activation of vitamin D 3 to calcitriol capable of promoting calcium absorption, and participate in blood-clotting cascade and muscular contraction 34 . Therefore, the concentration of Ca 2+ in the human body has become a vital index in research on tissue engineering and biomedicine 59 , increasing demand for Ca 2+ detection and Ca 2+ controllable release in interdisciplinary fields. In this context, our smart material may have potential applications in high-sensitivity Ca 2+ detection and portable devices in therapy, as well as promising Ca 2+ -actuated bioseparation membranes and microfluidic devices 60 .