HSA is the most prominent plasma protein in human blood, and it acts as a transporter or depot of insoluble endogenous and exogenous compounds such as fatty acids, hemin, bilirubin, thyroxine, metal ions, and a broad range of drugs.^36,37 Uranyl ion (UO₂²⁺), a widely used negative staining agent for transmission electron microscopy (TEM), strongly binds to the subdomain IIB of HSA.³⁸ The TEM observations of stained HSA NTs using uranyl acetate yielded positive images of the cylindrical walls (Figure 2F), which implied that UO₂²⁺ bonded to NTs.²⁵ Remarkably, the inner wall of the cylinder was clearly dark, which indicated that the sixth and most accessible layer was constituted of HSA. Moreover, we determined that a cyanine dye, Zn-porphyrin, and a fatty acid were also bound to the HSA components in the cylindrical wall of HSA NTs.²⁵

To promote the biospecific capturing ability of protein NTs, we introduced avidin (Avi, Mw: 68 kDa) as the last layer of the tubular wall. Avi from egg white binds four biotins with the highest affinity of any known protein (K > 10¹⁵ M⁻¹).³⁹ We prepared (PLA/HSA)₂PLA/PLG/Avi NTs (Avi NTs) using the same procedure (Figure 3A).²⁵ Because Avi is a basic glycoprotein (isoelectric point: 10.0–10.5) and has a positive net charge at pH 7.0, an anionic poly-l-glutamic acid sodium salt (PLG: Mw: ca. 70 kDa) was deposited on the fifth PLA layer to fix the final Avi layer. SEM images showed the formation of uniform cylinders with 403 ± 10-nm O.D., 59 ± 5-nm W.T., and ca. 9-µm T.L.

Figure 3. (A) Schematic illustration of bFNPs capture in an Avi NT. (B) Schematic illustration of HBVs trapping in HBsAb NT and TEM images of the NTs before and after virus capturing (scale bar: 200 nm). (C) Schematic illustration of CTC-E. coli entrapped fluorescent HSA MT and its CLSM image. Adapted with permissions from ref 25, 41, 48.

As a ligand for these hybrid protein NTs, we exploited a biotinylated fluorescein (bFL). The fluorescence intensity of an aqueous bFL solution disappeared after incubation with Avi NTs.²⁵ The bleaching of color was clearly observed, which indicated that bFL was bound to the Avi layer. The binding ratio of bFL and Avi was estimated to be ca. 3.4/1 (mol/mol). Biotinylated fluorescent nanoparticles (bFNPs) were also captured in the 1-D pore space interior of Avi NTs (Figure 3A).²⁵ The fluorescence intensity of the PB solution containing 100-nm bFNPs decreases after incubation with Avi NTs, which suggested that bFNPs were incorporated into the hollows. In contrast, a decrease in fluorescence was negligible after incubation with 250-nm bFNPs.²⁵ This difference showed that larger bFNPs could not enter the tubes because the swollen NT’s I.D. (ca. 200 nm) was smaller than the particle diameter. The average number of 100-nm bFNPs captured inside the tube were calculated to be 161. This suggests that the occupancy ratio of the particles in the volume was 29%.

Hepatitis B virus (HBV) infection is a serious global health problem. Despite the existence of vaccination, 350 million people suffer from chronic HBV worldwide.⁴⁰ Infectious HBV, so-called Dane particle (DP, 42-nm diameter), comprises a core nucleocapsid containing genome DNA covered with an envelope, which consists of a hepatitis B surface antigen (HBsAg, Mw: ca. 25 kDa). If one can synthesize a unique nanotubular trap for infectious DP, it would have an important impact not only on bioseparation chemistry but also on medical applications.

We prepared HSA-based NTs with an HBsAg antibody (HBsAb) as the last layer of the wall, using a nanoporous PC membrane (400-nm pore diameter), (PLA/HSA)₂PLA/PLG/HBsAb NTs (HBsAb NTs, Figure 3B).⁴¹ The dimensions of the tube were observed by SEM measurements (414 ± 16-nm O.D., 59 ± 4-nm W.T., and ca, 9-µm T.L.). To evaluate the DP-trapping capability of HBsAb NTs, DNA quantification assays of supernatants were conducted by PCR. As expected, DNA concentrations considerably decreases after incubation with HBsAb NTs.⁴¹ The trapping ratio reached 99.9%. We reasoned that DPs were perfectly ensnared into the 1-D pore space of NT. TEM observation demonstrated the entrapping of DPs into the hollow (Figure 3B). The removal efficiency by a single HBsAb NTs treatment reached a −3.9log order.

Another challenging target is influenza viruses. Among the three types of influenza viruses (A, B, and C), type A is a virulent pathogen that induces a severe disease.⁴² In 2009, a swine-origin influenza A (H1N1) virus spread worldwide and caused a human pandemic. The influenza A virus infection is initiated by the specific binding of viral hemagglutinin to a cellular surface receptor, containing sialyloligosaccharide with N-acetyl neuraminic acid (Neu5Ac) terminals.⁴³ Fetuin from fetal calf serum (Mw: 48.4 kDa) is a heavily glycosylated protein with tribranched oligosaccharides including Neu5Ac residues.⁴⁴ Influenza viruses are well known to interact with this natural glycoprotein.⁴⁵

We prepared HSA-based NTs with an innermost layer of fetuin using a nanoporous PC membrane (800-nm pore diameter), (PLA/HSA)₅PLA/fetuin NTs (Fetuin NTs).⁴⁶ SEM measurements demonstrated the formation of homogeneous hollow cylinders with 811 ± 13-nm O.D., 97 ± 3-nm W.T., and ca. 15-µm T.L. For trap experiments, we exploited the influenza A virus [Puerto Rico/8/34 (PR8, H1N1)] with a diameter of 94 ± 15 nm. The I.D. of Fetuin NTs is sufficiently large to accommodate PR8 virions. The virus trapping capability of NTs was examined by the direct enzyme-linked immunosorbent assay of the remaining PR8. The results showed that the PR8 solution treated with Fetuin NTs became completely virus-free.⁴⁶ We inferred that influenza A virus PR8 diffused into the hollow space of NTs and bonded to the inner surface wall on the basis of specific binding of hemagglutinin on the virus surface to the sialyloligosaccharide chain of fetuin. The removal efficiency by a single treatment with NTs was on the −5.0 log order.

These two astonishing results for HBsAb NTs⁴¹ and Fetuin NTs⁴⁶ provoke the development of a new field of nanometer-scale virus detection and removal devices. For example, the elimination of small viruses (hepatitis E viruses and human parvo B19 viruses) and corona viruses (SARS-CoV-2) that caused pandemic in 2020 would be of tremendous medical importance.

The next challenge is the trapping of a living organism. Escherichia coli (E. coli), a rod-shaped gram-negative bacterium (0.4–0.7-µm width, 2–4-µm length), is the smallest organism of the micrometer-scale world. Many strains are harmless, but some serotypes (e.g., enterohemorrhagic E. coli. O157) can induce severe poisoning in humans.⁴⁷ (PLA/HSA)₉ MTs (HSA MTs) were fabricated using a PC membrane (1.2-µm pore diameter).⁴⁸ SEM measurements revealed the formation of uniform hollow cylinders with 1.0 ± 0.02-µm O.D., 148 ± 5-nm W.T., and ca. 25-µm T.L.

Then, we measured the E. coli capture capability of HSA MTs. The dimensions of E. coli K12 were as follows, 425-nm width and ca. 2–3-µm length. The colony incidence of the sample solution after mixing with MTs was assayed using the standard plate count method. There were fewer colonies appearing on the plate (N_c) compared to those of identically treated E. coli without the tubes.⁴⁸ Surprisingly, N_c became completely zero after 30 min of mixing with HSA MTs; the disappearance yield reached 100%. However, incubation with slender HSA NTs exhibited no change in the colony number. We reasoned that E. coli entered the pores of swollen MTs (ca. 500-nm I.D.), but they were too large to enter the narrow NT’s channel (ca. 200-nm I.D.). The efficiency of removal by a single HSA MT treatment was over the −7log order.

The incorporation of E. coli into MTs was confirmed by confocal laser scanning microscopy (CLSM). The CLSM images of the mixture solution of fluorescein-labeled HSA MT and CTC (5-cyano-2,3-ditolyl tetrazolium chloride)-labeled E. coli demonstrated that CTC-E. coli (red) was loaded into the 1-D pore space of the MT (green) (Figure 3C).⁴⁸ It is remarkable that the edge of E. coli appeared from the mouth of the tube in most cases, thereby forming a “bacterium-corked MT”. Because the E. coli width is only slightly smaller than the pore size, it cannot enter deep inside the tubule.

Interestingly, E. coli trapped in the HSA MT was metabolically inactive. After 1 h of mixing, almost all E. coli lost cell proliferation ability.⁴⁸ The E. coli growth can be prevented by stopping cell deviation. In the narrow space of MTs, bacteria cannot reproduce through asexual reproduction by binary fission. This remarkable result will serve as a trigger to make novel bacteria removing devices.

HSA possesses a weak esterase activity for hydrolysis of 4-nitrophenylacetate (4NPA).³⁶ The reaction occurs in the subdomain IIIA of HSA. If HSA components in HSA NTs retain their original property, the tubules are also able to show the same enzyme activity. First, we evaluated an esterase activity of HSA NTs.³⁵ After adding 4NPA to the NT dispersion, absorbance increased at 440 nm, which was based on the product, 4-nitrophenol (4NP), absorption. Therefore, we suspected that 4NPA diffused into the cylindrical wall and was hydrolyzed to generate 4NP. The enzyme activity parameters [Michaelis constant (K_m) and catalytic constant (k_cat)] for the esterase activity of HSA NTs were similar to those of free HSA (Table 1).

Table 1. Enzyme activity parameters of protein MT and NT reactors

α-d-glucosidase from Saccharomyces cerevisiae (αGD, Mw: 68.5 kDa) is a typical exo-type carbohydrase, which is used to cleave an α(1→4) glucosyl linkage in the non-reducing terminal of the substrate.⁴⁹ We evaluated the enzyme activities of multilayered protein NTs with an αGD interior surface, (PLA/HSA)₂PLA/αGD NTs (αGD NTs) (413 ± 17 nm-O.D., 52 ± 3-nm W.T., and ca. 9-µm T.L.).⁵⁰ In an aqueous medium, NTs captured a fluorogenic glucopyranoside, 4-methylumbelliferyl-α-d-glucopyranoside (MUGlc), into their 1-D pore space and hydrolyzed the substrate with a release of α-d-glucose (αGlc). A non-competitive-type inhibition was observed in the enzyme parameters.⁵⁰ The probability of four subunits of adhered αGD facing toward the aqueous solution is half or one-fourth on the curved inner wall. This statistic geometry may be one of the reasons for the lower enzyme activity.

To elucidate the relation between enzyme activity and stratified wall structure (enzyme layer number and position) in detail, we prepared three different MTs, in which the GOD layer was introduced as the innermost layer, intermediate layer, or all-internal protein layers.⁵¹ (i) After the 7.5-cycle LbL growth of PLA/HSA, GOD was deposited onto the fifteenth PLA layer, which provided (PLA/HSA)₇PLA/GOD MTs (GOD1 MTs). (ii) GOD was introduced into the eighth layer, which yielded (PLA/HSA)₃PLA/GOD(PLA/HSA)₄ MTs (GOD2 MTs). (iii) Moreover, using GOD as all-internal protein layers produced (PLA/GOD)₇PLA/HSA MTs (GOD3 MTs). All tubules exhibited almost the same morphologies (ca. 1.2-µm O.D., ca. 135-nm W.T., and ca. 23-µm T.L.).

Upon the addition of β-d-glucose (βGlc) into the tube dispersion containing peroxidase and o-dianisidine, a color change was clearly visible, and absorbance at 460 nm increased.⁵¹ Three-types of MTs catalyzed the oxidation of βGlc to β-d-glucono-1,5-lactone and H₂O₂. We inferred that βGlc molecules diffused into the swollen tube wall and were oxidized by the GOD components. It is remarkable that the K_m and k_cat values of GOD1 MTs were equivalent to those of the GOD solution (Table 1).⁵¹ The immobilized enzymes on the interior surface wall retained their original catalytic activity. In contrast, non-competitive-type inhibition was observed in GOD2 and GOD3 MTs. A decrease in k_cat values is attributed to the low diffusion of the substrate in the cylindrical wall and blocked ligand sites by upper and lower PLA layers. We concluded that the performance of the innermost GOD layer was almost identical to that of a free GOD molecule; however, the catalytic activity of the intermediate layer declined by ca. 40–50% compared to that of monomeric GOD. Overall, enzymatic reactions, which occurred in protein MTs and NTs, are affected by several factors such as diffusion of substrates in the 1-D pore space and positions and geometries of the enzymes in layered walls.

There are no studies reporting enzymatic polymerization in NTs. If a nanotubular reactor for polymer synthesis were to be constructed, it would have a great impact on advanced material chemistry. Kobayashi et al. first reported ring-opening polymerization of lactones catalyzed by lipases in 1993.⁵² They determined that immobilized Candida antarctica lipase B (CalB, Mw: 33 kDa) was effective for polymerization.⁵³ We prepared HSA-based NTs with an inner wall composed of CalB, (PLA/HSA)₂PLA/CalB NTs (CalB NTs) (393 ± 9-nm O.D., 47 ± 5-nm W.T., and ca. 9-µm T.L.) and investigated their catalytic performance toward the ring-opening polymerization of lactone.⁵⁴ 12-Dodecanolactone (DDL) was added to an aqueous dispersion of CalB NTs. The mixture was continuously stirred at 22 °C for 24 h. Then, the resultant suspension was through to collect the white solid product. MALDI-TOF mass spectroscopy demonstrated the formation of oligomers with molecular weights of 638–2816.⁵⁴ The degree of oligomerization was calculated to be 3–14. Of note, the molecular weights of oligoDDL produced in aqueous CalB NTs were higher than those prepared in the CalB solution. The superior catalytic performance of CalB NTs may be due to the incorporation of a water-insoluble DDL monomer in the hollow. The contact area of the interface between the monomer droplet and the CalB wall increases in the cylinder, which accelerated the oligomerization process.

Metal NPs are also exploited as a layered wall component. Hybrid NTs combine the merits of both biomolecules and inorganic nanomaterials with the potential for widely diverse applications. We chose gold nanoparticles (AuNPs) because they have received a considerable interest owing to their catalytic activity as well as tunable optical and electronic properties.^55,56 Hybrid NTs were prepared by an alternate LbL assembly of a AuNP and HSA admixture ([HSA]/[AuNP] = (1 mg/mL)/(0.9 mg/mL) and PLA into a track-etched PC membrane (400-nm pore diameter) (Figure 4).⁵⁷ SEM images showed the formation of uniform hollow cylinders of (PLA/AuNP-HSA)₃ NTs (AuNP-HSA NTs) with 426 ± 12-nm O.D., 65 ± 7-nm W.T., and ca. 9-µm T.L.) (Figure 4A). TEM and energy dispersive X-ray (EDX) measurements supported the high loading of AuNPs in the tubular wall (Figure 4B).⁵⁷ The core–shell AuNP-HSA corona, in which the protein is bound to the NP surface, is essential for robust NT formation (Figure 4C). AuNP-HSA NTs serve as a heterogeneous catalyst for the reduction of 4NP with sodium borohydrate.⁵⁷ The superior performance of AuNP-HSA NTs is probably attributed to the more homogeneous distribution of AuNPs in the swollen HSA layers. We reasoned that HSA in NT equally dispersed AuNP units in the tube wall and concentrated the 4NP substrate in the cylinder from the bulk aqueous solution.

Figure 4. (A, B) (A) SEM and (B) TEM images of AuNP-HSA NTs. (C) Schematic illustration of AuNP-HSA NT. (D) SEM image of AuNP NTs (scale bar inset: 200 nm). (E) SEM image of AuNP-HSA NTs immobilized on the glass surface. Adapted with permission from ref 57.

Interestingly, the calcination of AuNP-HSA NTs at 500 °C in air yielded red solid NTs that were composed of thermally fused AuNPs.⁵⁷ The tubular structure remained intact, although the shrinkage of morphology was observed: 195 ± 10-nm O.D., 41 ± 4-nm W.T., and ca. 4-µm T.L. (Figure 4D). A detailed inspection revealed that the cylindrical wall consists of numerous AuNPs with a diameter of ca. 13 nm. AuNP NTs showed the same catalytic activity; however, the efficiency was lower than that of AuNP-HSA NTs.

Moreover, we demonstrated the template-assisted synthesis of AuNP-HSA cylinder arrays that were covalently attached to the glass surface.⁵⁷ Stiff AuNP-HSA NTs allowed us to create a “protein NT forest” on the planar substrate (Figure 4E). The NT density (7.9 × 10⁷ tubes/cm²) showed good agreement with the porosity number of the PC membrane (7.9 × 10⁷ pores/cm²).

Solid NTs comprising hematite nanoparticles (α-Fe₂O₃NPs) can be prepared from an iron-storage protein ferritin (Mw: 460 kDa).⁵⁸ The initial (PLA-ferritin)₃ NTs (Ferritin NTs) precursors were fabricated using a track-etched PC membrane (400-nm pore diameter). The obtained uniform cylinders (410 ± 14-nm O.D., 61 ± 5-nm W.T., ca. 9-µm T.L.) were calcined at 500 °C in air, which yielded reddish-brown iron oxide NTs. The hollow structure remained perfect, but its O.D., W.T., and T.L. were considerably smaller (211 ± 8 nm, 29 ± 3 nm, and ca. 5 µm, respectively). The disappearance of the protein shell and PLA layers was confirmed using infrared and EDX spectroscopies. Subsequent SEM, TEM, and X-ray photoelectron spectroscopy showed that tubular walls comprise fine α-Fe₂O₃NPs with a 5-nm diameter. Iron oxide NTs demonstrated superparamagnetic properties with a blocking temperature of 37 K and efficient photocatalytic activity for the degradation of 4-chlorophenol.⁵⁸

Self-propelled MT motors have attracted considerable interest during the last decade.^59–69 Many metal-based capillaries containing an internal wall of a Pt thin film have been fabricated by rolling-up processing with photolithography or templating synthesis with electrochemical deposition. A disproportionation reaction of H₂O₂ (2H₂O₂ → 2H₂O + O₂) occurs on the Pt surface with the subsequent continuous ejection of O₂ microbubbles from the terminal opening. Thus, the tube gains thrust in the direction opposite to the bubble discharge. The O₂ bubble expulsion is responsible for autonomous propulsion. These swimming MTs have been demonstrated to be useful for environmental applications^63,64,66 such as pollutant cleaners and separation devices. Other goals of tubular motor development are practical life science applications for human health^67–69 such as on-demand vehicles for targeted drug delivery and elements of microdiagnostic devices. These smart MTs must have biocompatibility, which is the most important feature. However, the inorganic rigid micropipes with Pt walls are not biodegradable. As an alternative, citrate-stabilized Pt nanoparticles (PtNPs) have been widely used in the medical field as a strong quencher for O₂^•− and H₂O₂.⁷⁰ The PtNP itself shows almost no cytotoxicity.⁷¹ We prepared HSA-based MTs with an interior surface composed of PtNPs by templating synthesis using a track-etched PC membrane (1.2-µm pore diameter), (PLA/HSA)₈PLA/PtNP MTs (PtNP MTs).⁷² SEM and TEM observations revealed the formation of uniform hollow cylinders (1.16 ± 0.02-µm O.D., 147 ± 11-nm W.T., and ca. 23-µm T.L.) (Figures 5A, B). In aqueous H₂O₂ media (pH 7.0, 5-wt % H₂O₂, 1-wt % SDS). PtNP MTs are self-propelled by jetting O₂ bubbles from the open-end terminus (Figure 5C, Movie S1).⁷² The disproportionation of H₂O₂ was catalyzed by the innermost PtNP layer. The continuous O₂ bubble releases from one end induced self-pumping of peroxide fuel in the hollow space. The resulting liquid flow in the capillary propels the tube (Figure 5C). The swimming trajectories of PtNP MTs mostly show a turning motion, which is attributable to the asymmetrical shape of the tube’s body and mouth. The PtNP MTs velocity depended on the H₂O₂ concentration of 1–10 wt %, which typically was 273 ± 26 µm/s (12.3 body lengths/s) in 5-wt % H₂O₂. By adding a magnetite nanoparticle (Fe₃O₄NP) layer in the tube wall, the direction of MT movement can be manipulated to become a straight motion using magnetic field guidance.⁷²

Figure 5. (A, B) (A) SEM and (B) TEM images of PtNP MTs. (C) Micrographs taken from Movie S1 of microscopic observation of self-propelled PtNP MT by jetting O₂ bubbles from the open-end terminus in a PB solution (pH 7.0, 5-wt % H₂O₂, 1-wt % SDS) and the schematic illustration of a swimming PtNP MT. Adapted with permission from ref 72.

Because the exterior surface of MTs is positively charged, negatively charged bacteria would be adsorbed on the tube. We exploited a genetically engineered E. coli in which green fluorescent protein (GFP) was secreted. Fluorescent E. coli was added to the self-propelled PtNP MT dispersion. PtNP MTs swam in the E. coli suspension by spouting out O₂ microbubbles. After 15 min, the tubules were collected, and the remaining free E. coli was lysed to obtain a clear GFP solution. The fluorescence intensity of GFP in the lysate was markedly low (only 1%) compared to the value of the identically treated lysate without the tubes.⁷² The 99% removal of E. coli was achieved by single treatment with self-propelled MTs. The moving motion is essential for accelerating the contact frequency to catch the bacteria. Our results showed that swimming MTs captures E. coli efficiently and function as an “automatic micromop”.

To provide additional functionality to swimming MTs, it is required to use the outer surface of the cylinder. Nevertheless, a shortcoming of the wet-template synthesis lies in the difficulty of changing the exterior surface. The outside wall, which is composed of the initial filtration materials, must be PLA in our procedure. To overcome this difficulty, we produced an electrostatic LbL coating of released MTs with Avi protein in bulk aqueous solution.⁷³ Avi–biotin complexation allows swimming MTs to capture various biotinylated substances.

We dispersed PtNP MTs in a PB solution and electrostatically wrapped them with Avi by LbL coating, which yielded Avi/PLG(PLA/HSA)₈PLA/PtNP MTs (Avi/PtNP MTs, ca. 1.2-µm O.D. and ca. 24-µm T.L.).⁷³ Avi/PtNP MTs were self-propelled in an H₂O₂ solution by the ejection of O₂ bubbles with an average velocity of 141 ± 8.7 µm/s (5.9 body lengths/s) (pH 7.0, 5-wt % H₂O₂, 0.2-wt % Triton X-100).

To confirm the exterior surface of Avi and to evaluate the selective biotin binding ability, we added biotinylated fluorescein (bFL) to the PB solution of the tubes. Swimming Avi/PtNP MTs could efficiently capture bFL and strongly fluoresced.⁷³ The swimming motion can be observed using optical microscopy under fluorescence mode (Figure 6A, Movie S2). Even with the coexistence of different dyes, bFL was specifically captured by Avi/PtNP MTs.

Figure 6. (A) Micrographs from Movie S2 of microscopic observations of a bFL-bound self-propelled Avi/PtNP MT jetting O₂ bubbles in a PB solution (pH 7.0, 5-wt % H₂O₂, 0.2-wt % Triton X-100) under fluorescence mode. (B) Schematic illustration of bFNP15 and bαGD binding to the exterior surface of Avi/PtNP MT. Adapted with permission from ref 73.

The next challenge is the transportation of nanometer- and micrometer-size particles. Upon addition of biotinylated fluorescent nanoparticles with a diameter of 15 nm (bFNP15) to the PB solution of swimming Avi/PtNP MTs, the particles immediately adhered to the tubes’ exterior surface (Figure 6B).⁷³ Swimming bFNP15-bound Avi/PtNP MTs emitted red fluorescence. The larger bFNP100s (100 nm) and bFMP4s (4 µm) were also caught by the tubular motors.⁷³

Enzyme immobilization on the outer surface would allow swimming tubes to have a biocatalytic activity. Its self-propulsive motion is expected to facilitate the enzyme reaction. We again used the αGD enzyme. Biotinylated αGD (bαGD) was added to the PB solution of Avi/PtNP MTs yielding αGD-covered Avi/PtNP MTs [bαGD/Avi/PLG(PLA/HSA)₈PLA/PtNP MTs (bαGD-Avi/PtNP MTs)] (ca. 1.2-µm O.D. and ca. 24-µm T.L.) (Figure 6B).⁷³ After the addition of MUGlc to the swimming tube dispersion, the blue fluorescence of the product, 4-methylumbelliferon (MU), was visible. The enzyme activity of bαGD-Avi/PtNP MT motors was approximately 30% of that of free bαGD.⁷³ This restricted activity likely occurred because αGD molecules densely adhered to the exterior surface with statistic geometries. The result is consistent with that of αGD NTs with an enzyme interior surface.⁵⁰ Our results demonstrated that self-propelled protein MTs act as ultrasmall transporters, removers, separators, and stirrers.

Catalase (Cat, Mw: 240 kDa) is a common hemoprotein enzyme that is capable of decomposing H₂O₂ with high efficiency in antioxidant defense systems. Several successful experiments related to Cat-powered polymer MTs have been reported,^74–76 although they still show poor biocompatibility. If one were able to immobilize Cat on the inner surface of protein MTs, the obtained cylinders could possess both self-propulsion capability and biodegradability. We prepared protein MT motors containing Cat as an interior surface, (PLA/HSA)₇PLA/PLG/Avi/bCat MTs (Cat MTs).⁷⁷ Unfortunately, the direct LbL synthesis of designed tubes failed. Therefore, we employed a two-step preparation using Avi–biotin interaction with biotinylated Cat (bCat). Optical microscopy revealed that the O.D. and T.L. of Cat MTs were ca. 1.2 µm and ca. 24 µm, respectively.

Upon addition of H₂O₂ into the aqueous Cat MT dispersion, the tubes were self-propelled by jetting O₂ bubbles from the terminal opening with the average velocity of 71 ± 20 µm/s (3.0 body lengths/s) (pH 7.0, 5-wt % H₂O₂, 0.2-wt % Triton X-100) (Figures 7A, B, Movie S3A).⁷⁷ Azide anion is known to coordinate to the heme groups of Cat and inhibit enzyme activity. Actually, the addition of NaN₃ stopped the movement of tubes. The cylindrical wall of protein multilayers must be soft, although the MT structure was sufficiently stable to endure the bubble-propelled motion in water.

Figure 7. (A) Micrographs taken from Movie S3A of microscopic observations of a self-propelled Cat MT by jetting O₂ bubbles in the PB solution (pH 7.0, 2-wt % H₂O₂) (scale bar, 20 µm). The bottom image (enlarged image) shows a line of moving O₂ bubbles in the capillary (red arrow). (B) Schematic illustration of swimming Cat MT with O₂ bubble ejection from the terminal opening. (C) Slow motion images of O₂ bubble migration in MT and their expulsion from the tube mouth taken from Movie S3B (scale bar, 10 µm). (D) Effect of pH on the average velocity of self-propelled Cat MTs (5-wt % H₂O₂, 0.2-wt % Triton X-100) and on the Cat activity⁷⁸ at 25 °C. Adapted with permission from ref 77.

To our surprise, the migration of O₂ bubbles was readily visible at the center of the capillary because protein walls are transparent and reasonably long (Figure 7A bottom, Movie S3B).⁷⁷ Careful inspection revealed that two expulsions every 45 ms (22.2 Hz) from the tube’s mouth create one bubble in the outer aqueous phase (Figure 7C). For example, the first expulsion at 5 ms created one small bubble (ca. 3-µm diameter) in the outer aqueous phase (red arrow). The second expulsion at 50 ms enlarged this bubble to 5 µm. The third expulsion at 90 ms released the bubble (red arrow) and generated another new one (light blue arrow).

The pH and temperature greatly affect the enzyme activity. Indeed, the swimming speed of Cat MTs was affected by pH (pH 6.0–9.0). It is particularly interesting that the pH dependence of velocity exhibited a maximum peak at pH 7.0, which corresponded to the optimum pH of Cat (Figure 7D).^77,78 As expected, the temperature dependence of the tube’s speed (15–50 °C) also coincided with the Cat activity curve.⁷⁷ We concluded that the Cat MT motor velocity could be regulated by modulating enzyme activity with pH and temperature.

Cat MTs consist only of proteins and polypeptides. Therefore, the tubular wall can be easily decomposed by acid and protease. By adding Pronase (a cocktail of several proteases) into the Cat MT dispersion, the hollow column morphology completely disappeared.⁷⁷

Cat enzyme is a promising alternative to inorganic catalysts. However, some concerns still remain. (i) The H₂O₂ fuel is a powerful oxidative reagent that is toxic to cells and tissues. (ii) Gas bubble formation may be undesirable in an in vivo environment. Recently, Sánchez et al. have reported that silica NTs with an internal wall bearing urease (Ure) were self-propelled in water without bubble spouting.⁷⁹ The NTs (220-nm O.D.) were driven by the turnover of the urea substrate triggered by the Ure reaction (Urea + H₂O → 2NH₃ + CO₂). Urea is essential in the metabolism of nitrogen-containing compounds by animals. The normal range of urea is 2.1–7.1 mM in human blood and 100–450 mM in human urine.⁸⁰

Using Jack bean Ure (Canavalia ensiformis, Mw, 480 kDa), we prepared HSA-based MTs containing a Ure inner wall, (PLA/HSA)₇PLA/PLG/Avi/bUre MTs (Ure MTs).⁸¹ Optical microscopic observations demonstrated that the homogeneous cylinder structure was preserved after biotinylated Ure (bUre) binding (ca. 1.2-µm O.D. and ca. 24-µm T.L.). Ure MTs swam very smoothly with nonbubble propulsion in a PB solution containing physiological concentration of urea (100 mM) (Figure 8, Movies S4).⁸¹ The swimming trajectory of the tube showed a turning motion (Figure 8A, Movie S4A) or a straight motion (Figure 8B, Movie S4B). The average velocity was 2.7 ± 0.2 µm/s. The decomposition of urea occurs on the innermost Ure layer. The products NH₃ and CO₂ are diffused from the 1-D pore space to the bulk aqueous solution (Figure 8C). This diffusiophoresis may be responsible for autonomous propulsion. In addition, Ure MTs entirely disappeared in the aqueous solution of protease, which shows their adequate biodegradability.⁸¹

Figure 8. (A, B) Micrographs taken from Movies S4 of microscopic observations of self-propelled Ure MTs in the PB solution (10 mM, pH 6.0) containing 100-mM urea at 25 °C. The times shown in these figures are actual times. The video speeds in Movie S4 are fast-forwarded to 2.4-times the actual speed. (A) Turning motion shown in Movie S4A. (B) Straight motion for 30 s with trajectory (red line) from Movie S4B. (C) Schematic illustration of a swimming Ure MT motor with nonbubble propulsion. Adapted with permission from ref 81.