Volume 50 Number 68 4 September 2014 Pages 9613–9784

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Chemical Communications
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T. Komatsu et al.
An Escherichia coli trap in human serum albumin microtubes

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An Escherichia coli trap in human serumalbumin microtubes†
S. Yuge, M. Akiyama and T. Komatsu*

Received 14th May 2014,Accepted 10th June 2014
DOI: 10.1039/c4cc03632h
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We describe the template synthesis of human serum albumin microtubes (MTs) and highlight their Escherichia coli (E. coli) trapping
capability with extremely high eﬃciency. The E. coli was loaded into
the one-dimensional pore space interior of the tubule. Similar MTs
including an Fe3O4 layer also captured E. coli and were manipulated
by exposure to a magnetic field.

Hollow, cylindrical, nanometer-scale structures comprising biomaterials, i.e. bionanotubes, have attracted considerable attention
because of their potential applications as molecular trapping
devices, drug delivery containers, and enzymatic reactors.1–13
Alternate layer-by-layer (LbL) build-up assembly of proteins,4,6,7,9,10
DNAs,5,8,11 and antibodies12,13 in nanoporous membranes enables
creation of structurally defined smart nanotubes (NTs) withversatile biochemical reactivities. The one-dimensional (1D) porespace interior of the tubule can be tailored by deposition of adesired material. Therefore, many investigators have exploredthe loading of nanometer-size entities into the channel, such as
pharmaceutical drugs,9,12 bioactive spheres,9 inorganic colloids,11and infectious viruses.13 Another challenging subject in this fieldwould be the trapping of a living organism. Escherichia coli
(E. coli), a rod-shaped gram-negative bacterium, is the smallest
organism of the micrometer-scale world (0.4–0.7 mm width,2–4 mm length). Many strains are harmless, but some serotypes cancause severe poisoning in humans, such as enterohemorrhagic
E. coli. O157.14 If one were able to generate a unique E. coli trap
in protein microtubes (MTs), then it would have an important
contribution not only to bioseparation chemistry, but also todiverse aspects of human health. This communication is thefirst to describe the synthesis and structure of human serum
albumin (HSA)-based MTs, and to highlight their excellent
E. coli trapping capability.
Department of Applied Chemistry, Faculty of Science and Engineering,
Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan.
E-mail: komatsu@kc.chuo-u.ac.jp† Electronic supplementary information (ESI) available: Experimental section,
the SEM image of E. coli, the CLSM image of CTC-E. coli, and the relationshipbetween mixing time and cell viability of E. coli. See DOI: 10.1039/c4cc03632h

9640 | Chem. Commun., 2014, 50, 9640--9643

The MTs were fabricated by template synthesis using electrostatic LbL assembly.9 Briefly, positively charged poly-L-arginine(PLA) and negatively charged HSA were alternately deposited(nine-cycles) onto the pore wall of a track-etched polycarbonate(PC) membrane (1.2 mm pore size). Subsequent dissolution of thePC framework in N,N-dimethylformamide and freeze-drying of theliberated core yielded (PLA/HSA)9 MTs as white powder. SEM
measurements revealed the formation of uniform hollow cylinders
with 1.0  0.02 mm outer diameter and 148  5 nm wall thickness(Fig. 1a and b). The tube lengths (ca. 25 mm) coincided well with thePC membrane pore depth. In contrast, 12-layered (PLA/HSA)6 MTs
were wrinkled and fragile, which implies that six-cycle injection
is insuﬃcient to generate stiﬀ MTs. We concluded that at leastnine-cycle deposition is necessary to construct a robust 1 mm-width
cylinder by our template synthesis. Based on the general principleof LbL membrane growth, we hypothesized a nine-layered cylindermodel. The average thickness of a PLA/HSA bilayer in the MT isestimated to be 16.4 nm. If one postulates the dimensions ofHSA to be 8 nm from the data of the single-crystal structure15
and small-angle X-ray scattering analyses,16 then the PLA layer
thickness is calculated to be 8.4 nm, which is between thereported values for typical polyelectrolyte layers prepared in theporous template by a wet process.17–19
The obtained (PLA/HSA)9 MTs were dispersed in deionized water,yielding a slightly turbid solution. To evaluate the morphology andstability of the MTs in water, the aqueous dispersion was freezedried in vacuo. SEM images demonstrated that the tubular walls
swelled considerably and that their thickness became 250  7 nm inwater (Fig. 1c). It is interesting that the outer diameter was unaltered(1.0  0.04 mm). Consequently, the inner pore size diminishedto ca. 500 nm. The average thickness of an individual PLA/HSAbilayer was measured to be 28 nm in water. Under the assumption that the HSA size did not change (r8 nm),15,16 the PLA
layer thickness might be 20 nm. This value is apparently larger
than that of the dry form, but it is similar to the values of thepolyelectrolyte layers in the NTs prepared under pressure
conditions.9,20 We determined the swelling ratio of the PLA
layer (aPLA: a ratio between the section area in the swollen stateThis journal is © The Royal Society of Chemistry 2014View Article Online

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CommunicationFig. 1 SEM images of (PLA/HSA)9 MTs prepared using a porous PCtemplate (Dp = 1.2 mm): (a, b) dried samples and (c) lyophilized sample
after swelling in water. (d) SEM image of Fe3O4(PLA/HSA)9 MTs preparedusing a porous PC template (Dp = 1.2 mm). (e) Schematic illustration ofE. coli entrapped (PLA/HSA)9 MTs.

and that in a dried state) to be 2.1 (see ESI†), which is almost
identical to the data reported in the literature for generalpolyelectrolytes (1.2–4.0).21,22 Morphologies of the (PLA/HSA)9
MTs were retained for more than 24 h in water at 25 1C.
Then we measured the E. coli capture capability of the (PLA/HSA)9
MT. The dimensions of E. coli K12 (XL10-gold) we used were 425 nm
width and approximately 2–3 mm length, as revealed by SEM measurements (Fig. S1, ESI†). The colony incidence of the sample solution
after mixing with the MTs was assayed using a standard plate countmethod. First, E. coli (1  108 CFU mL 1, 100 mL) was added to theaqueous solution of (PLA/HSA)9 MTs (ca. 150 mg mL 1, 900 mL).23
The resultant mixture was incubated with gentle rotation at 25 1C for
1–30 min. Then a part of the solution was spread on the LB agar plateand cultured at 37 1C for 16 h. The colonies appearing on the plate[Nc(MT)] were markedly fewer than those of identically treated E. coli
without the tubes [Nc(control)]. To our surprise, Nc(MT) became
completely zero by 30 min mixing with the MTs (Fig. 2a and b);
the disappearance yield (100 – colony incidence) reached 100%.
Incubation with the 6-layered thin (PLA/HSA)3 nanotubes (NTs)(ca. 200 nm inner diameter) exhibited no change in the colony
number. We reasoned that the E. coli (425 nm width) entered thepore of the MT (ca. 500 nm), although it is too large to enter thenarrow NT’s channel (ca. 200 nm).
The incorporation of E. coli into the MT was proved using
confocal laser scanning microscopy (CLSM). To visualize the tube,This journal is © The Royal Society of Chemistry 2014

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Fig. 2 (a) Appearance of LB agar plates spread with E. coli dispersion
which was incubated with (PLA/HSA)3 NTs or (PLA/HSA)9 MTs for 30 min(after 16 h cell culture at 37 1C). (b) Relationship between mixing time andcolony incidence [Nc(NT) or Nc(MT)/Nc(control)  100] (n = 3).

a fluorescein-labeled HSA (f-HSA) was exploited as an intermediate
layer component, yielding (PLA/HSA)7PLA/f-HSA/PLA/HSA MTs(fluorescent MTs). E. coli was also stained with 5-cyano-2,3ditolyl tetrazolium chloride (CTC). This labeling agent specifically stained the edge of the E. coli rod (Fig. S2, ESI†), which isquite helpful to distinguish the E. coli position in the tube. In
CLSM images of the mixture solution, the tube wall fluoresced
green (Em. 522 nm); the hollow structure was clearly visible.
CTC-E. coli also showed sharp fluorescence in red (Em. 630 nm).
Overlapping these pictures with a DIC image demonstrates that
CTC-E. coli was loaded into the 1D pore space of the MT (Fig. 3).
Remarkably, the edge of E. coli appeared from the mouse of theFig. 3 Overlapping images of DIC and CLSM of fluorescent MTs incorporating
CTC-E. coli. (a) CTC-E. coli remained at the terminal of the tube and (b) small
CTC-E. coli reached the depth of the tube. Ex. 488 nm.

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ChemCommCommunicationFig. 5 (a) Photograph of collection of E. coli-loaded Fe3O4(PLA/HSA)9
MTs by a magnetic field. (b) Appearance of LB agar plates spread with
E. coli dispersion which was incubated with Fe3O4(PLA/HSA)9 MTs for
30 min (after 16 h cell culture at 37 1C).
Fig. 4 Relationship between mixing time and colony incidence of E. coli(n = 3).

tube in most cases, thereby forming ‘‘bacterium-corked MT’’(Fig. 1e and 3a). The E. coli width is only slightly smaller than thepore size (ca. 500 nm). Therefore it cannot enter deep inside ofthe tubule. Sometimes, a few E. coli are able to diﬀuse into thechannel and reach the tube depth (Fig. 3b).
Does E. coli really like the 1D hollow microspace with highdensity of HSA? To make this point clear, we prepared comparable
MTs with diﬀerent interior surfaces using poly-L-glutamic acidsodium salt (PLG) and poly(sodium 4-styrenesulfonate) (PSS) (bothnegatively charged polymers) instead of the last layer of HSA. SEM
measurements revealed that the (PLA/HSA)8PLA/PLG MTs and(PLA/HSA)8PLA/PSS MTs possess the same structures (outer/innerdiameters and length) as (PLA/HSA)9 MTs. Predictably, these MTs
without the HSA pore wall could not capture E. coli (Fig. 4). We
inferred that the inner surface wall of the tube must be comprisedof HSA to entrap E. coli. HSA can reversibly bind many bacterial
species by interacting with expressed surface proteins.24 Bacteria
move in response to a chemical stimulus, i.e. chemotaxis. For
instance, E. coli directs its movements according to certain chemicals in the environment.25 We reasoned that multiple factors
contribute to the E. coli capture into the (PLA/HSA)9 MT.
To clarify the fate of the entrapped E. coli, the cell viability wasmeasured using WST assay. It is noteworthy that E. coli accommodated in the (PLA/HSA)9 MT was metabolically inactive. After
1 h mixing, almost all E. coli lost cell proliferation ability (Fig. S3,ESI†). One possible explanation is that E. coli growth might beblocked by stopping cell deviation. In the narrow space of theMT, bacteria cannot reproduce through asexual reproduction bybinary fission. Another proposed mechanism is degradation ofthe HSA wall by OmpT, which is an aspartyl protease found on
the outer membrane of E. coli.26 This protease on the E. coli’s
surface comes into contact with the HSA inner-wall and cleaves
the polypeptide. Subsequently, the exposed cationic PLA layer
might cause cytotoxicity.
Moreover, we introduced a magnetite (Fe3O4) nanoparticle
layer into the MT. Magnetic-field assisted bioseparation using
spherical particles including Fe3O4 has been an area of particular
recent interest because of its diverse medical applications.27,28

9642 | Chem. Commun., 2014, 50, 9640--9643

Nevertheless, few reports describe the use of hollow magnetic
cylinders for bioseparation.12,17,29 An HSA-based MT bearing aferrimagnetic layer would become a magnetically responsive trap forE. coli. The magnetic tubes were prepared using a similar LbL
assembly procedure with Fe3O4 nanoparticles. SEM observations ofthe Fe3O4(PLA/HSA)9 MTs revealed the formation of highly ordered
arrays of the MTs with an outer diameter of 1.0  0.03 mm andthe maximum length of ca. 25 mm (Fig. 1d). The wall thickness
was 155  7 nm, which is slightly thicker than that observed inthe (PLA/HSA)9 MTs.
Finally, the E. coli capture ability of these magnetic MTs
was evaluated. As expected, the Fe3O4(PLA/HSA)9 MTs canbe collected by exposure to a magnetic field. By bringing aneodymium magnet close to the quartz cuvette including theE. coli solution with the Fe3O4(PLA/HSA)9 MTs, the brown tubes
were attracted rapidly to the magnet; the solution became colourless (Fig. 5a). Detaching the magnet from the cuvette liberated theMTs in the aqueous phase. This magnetic-field-induced collection–
dispersion was observed to be reversible. Then a part of the upper
clear solution in a cuvette, in which the E. coli loaded-Fe3O4(PLA/HSA)9 MTs were gathered at the bottom by the magnetic field,
was spread on an LB agar plate and cultured at 37 1C for 16 h.
The number of colonies appearing on the plate became zeroafter 30 min mixing with Fe3O4(PLA/HSA)9 MTs (Fig. 5b). The
disappearance yield reached 100%. We concluded that theE. coli entered the channel of Fe3O4(PLA/HSA)9 MTs in a similarfashion to that of (PLA/HSA)9 MTs.
In conclusion, the blood serum protein HSA microtubes
ensnared the E. coli perfectly. The eﬃciency of removal by a single
treatment with (PLA/HSA)9 MTs was over 7 log order. This remarkable result will serve as a trigger to produce a new and productive
field of removing device systems for bacteria. For example, elimination of enterohemorrhagic E. coli O157, using (PLA/HSA)9 MTs, is
expected to be of incredible medical importance. Furthermore,
E. coli-loaded MTs containing an Fe3O4 layer were magneticallymanipulated in solution. Recombinant HSA is currently manufactured on an industrial scale,30 which enables production ofthese protein MTs for practical use.
This work was supported by a Grant-in-Aid for Scientific
Research on Innovative Area ‘‘Coordination Programming’’(Area 2107, No. 21108013) from MEXT Japan, Grant-in-Aid forThis journal is © The Royal Society of Chemistry 2014

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Challenging Exploratory Research (No. 26600030) from JSPS,
and a Joint Research Grant from the Institute of Science andEngineering, Chuo University.

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