Focusing Review

CHROMATOGRAPHY, Vol.31 No.1 (2010)

Focusing Review

Development of high-performance analytical methods using
biomolecules encapsulated in silicate nanomaterials.
Kumiko Sakai-Kato
Division of Drugs, National Institute of Health Sciences
1−18−1 Kamiyoga, Setagaya-ku, Tokyo 158−8501, Japan

Abstract
High-throughput assay system using immobilized biomolecules with high accuracy and sensitivity would make drug discovery more efficient.
To utilize the biomolecules into analytical systems, we created immobilization techniques of biomolecules, especially proteins, using hydrogels derived from sol-gel reaction of alkoxysilane or silica nanoparticles. The silicate formed networks around proteins, and then gelled, effectively entrapping the proteins. We analyzed in detail the network patterns formed by the silica nanoparticles by means of the high-resolution
transmission electron microscopy. Furthermore, FT-IR study indicated immobilized proteins maintained their structure even after encapsulation in the gel. This immobilization technique enabled the integration of biochemical reaction, separation, and detection into capillary electrophoresis, capillary electrochromatography, microchip electrophoresis, and biochip systems.
Key Words: Sol-gel, Alkoxysilane, Silica nanoparticle, Biomolecule

ideal materials for bio-immobilization because this process can be

1. Introduction
Recently, genomic drug discovery technology, organic syn-used for many of the immobilization methods mentioned above andthesis, and in silico drug design technology have been used to de-because it can generate various matrix forms. In addition, thevelop molecularly targeted medicines [1, 2]. Under these condi-physical and chemical properties of silica are quite attractive fortions, high-throughput assay system with high accuracy and sensi-

joining inorganic materials with biomolecules.

tivity would make drug discovery more efficient. Biomaterials, asWe developed the methodology for bio-immobilization andtypified by proteins, serve various biological functions including

the application of sol-gel technology to analytical devices, espe-

molecular recognition, binding, catalysis, molecular transport, andcially microfluidic flow-through systems: capillary electrophoresis,

signal transduction. These functions are extremely accurate and ef-capillary electrochromatography, microchip electrophoresis, andficient owing to their conformational accuracy and diversity.

biochip systems; to achieve rapid analysis, reduced sample con-Therefore, the assay system using biomolecules is very attractive

sumption and cost [4, 5]. Furthermore, the specific and efficientfor drug discovery.

biological reactions may benefit greatly with microfluidic devices
that offer the means for handling small-volume samples (i.e., nL to

The various bio-immobilization methods reported to date in-µL) in confined reaction zones.

clude covalent attachment to supports, adsorption onto solid supports, cross-linking with bifunctional reagents, entrapment in gels,

2. The Development of Sol-gel Technology using Alkoxysilanes

and encapsulation in membranes such as microcapsules, liposomes,
hollow fibers, or dialysis membranes [3]. Silica matrices produced

The basic sol-gel process consists of the following steps: (1)by means of the sol-gel process have attracted much attention ashydrolysis of an alkoxysilane, (2) dimerization of the resulting sila-Correspondence: Division of Drugs, National Institute of Health Sciences, 1−18−1 Kamiyoga, Setagaya-ku, Tokyo 158−8501, Japan
Tel/Fax: +81−3−3700−9662
E-mail: kumikato@nihs.go.jp―１７―

CHROMATOGRAPHY, Vol.31 No.1 (2010)

nol to form siloxane bonds (≡Si−O−Si≡); and (3) polycondensa-

In CEC, the mobile phase contains an electrolyte and is trans-

tion reactions that link additional silanol molecules to form cyclic

ported through the capillary column by electroosmotic flow (EOF).

oligomers (Fig. 1). The most common starting materials are te-

The EOF is generated at the interface between the solution and atramethoxysilane (TMOS) and tetraethoxysilane (TEOS) because

charged surface, such as the deprotonated silica [14]. The sol-gelsthey undergo hydrolysis and condensation readily under mild con-also possess some unreacted silanols, which are believed to be re-ditions.

sponsible for the resultant EOF. Attenuated Total Reflection (ATR)-FT-IR was used to measure the silanol content during the time-

Elleby et al. have developed a modified version of the processcourse of gelation.

that removes the need for the addition of methanol. This version
does not expose the biomolecules to the damaging effects of lowFig. 2 a shows a typical IR spectrum of the gel in the region

pH [6]. These improvements enabled encapsulated proteins to re-between 1300 cm−1 and 800 cm−1. The band at 960 cm−1, indicatedtain their structure in nm-size porous structure [3, 6, 7] and biologi-

by number 1, corresponds to silanol groups. The band at 1016 cm−1,cal activity for a prolonged period [8] and to enhance its utility asindicated by number 2, corresponds to MeOH, which was gener-

new type of matrix for biomolecules. However, the uses of the re-ated in the process of hydrolysis and condensation. The bands indi-vised version had been limited to static formats, including silica-gel

cated by 3-6 correspond to SiO2 structure with different vibration

film [9] or monoliths in cuvettes for optical biosensors [6, 8, 10]
and it had not been applied to the flow-through supports for proteins, except for immunoaffinity chromatography in which IgGencapsulated sol-gel glass was ground and packed into a column[11].
Based on the Ellerby’s method, we developed a novel sol-gel
method for monolithic capillary columns that encapsulate proteins.
The greatest challenge in the design of the continuous gels was tofind controlled conditions showing the suitable characteristics aschromatographic packing material, that is, the proper mechanicalstrength and flow penetration. The properties of sol-gel matrices
depend on the relative rates of hydrolysis and condensation, which
can be altered by varying the pH, temperature, reagent concentrations, and reaction time [12, 13].
First of all, we searched for the optimal gelation condition
with no use of proteins, by varying buffer concentration, pH, andvolume. After that, other factors were examined by dissolving proteins in the buffer.

Fig. 2 Effect of residual silanol groups on EOF.
(a) Deconvolution of the IR absorption band at 800−1300 cm−1 ofhydrogel. Solid line: experimental spectrum. Dashed line: decomposed spectrum. Dotted line: sum of the six decomposedspectra. Sample: hydrogel fabricated using 5 mM phosphatebuffer.
(b) Relationship between the electroosmotic mobility and the relative contents of silanol groups. CEC conditions: Sample: 5 mMthiourea. Mobile phase: phosphate buffer. Applied voltage: 4kV. Injection: 4 kV, 5 s. Column length: 36 cm. Gel length: 5cm. Detection: 254 nm.

Fig. 1 Typical sol-gel reaction using alkoxysilane.

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Kumiko Katomodes. The peak area of the silanol peak gradually decreased andstructure and reactivity of the encapsulated biomolecules. Our re-the peak area of SiO2 gradually increased, as the gelation processsults support that this encapsulating technique is very effective toprogressed.

make use of biomolecules in biological assay system.

As shown in Fig. 2 b, the residual silanol groups decreased as
3. Application to analytical systems using CE or CEC

buffer pH of gel preparation increased. This change coincided withthat of electroosmotic mobility. The residual silanol groups also

The analytes of our assay system are mainly small molecules

decreased with an increase in the ionic strength, which is associ-and they can come in the silica networks and interact with the im-ated with the decrease in the electroosmotic mobility. These results

mobilized biomolecules. Two chiral compounds, bovine serum al-indicate that electroosmotic mobility have close relationship withbumin (BSA) and ovomucoid (OVM) from chicken egg white,

the content of residual silanol groups in gels [13]. Therefore, EOF

were encapsulated in tetramethoxysilane (TMOS)-based hydrogel

can be controlled by changing the gelation conditions. Of course,

and their chiral selectivity was evaluated for the separation of someother factors such as mobile phase conditions largely influence

selected enantiomers (tryptophan, benzoin, eperisone and chlor-electroosmotic mobility [12] .

pheniramine). Two different proteins, BSA and OVM, were suc-

IR spectra also give a variety of information on the protein

cessfully encapsulated into the gel matrices by changing the alkox-conformation [15]. The conformation of BSA in gel and in free so-

ysilane compositions of the gel and could separate the enantiomers

lution was investigated using ATR-FT-IR. As illustrated in Fig. 3,(Fig. 4) [12]. Run-to-run repeatability was quite satisfactory. The

two representative bands originated from BSA were observed inconsecutive analysis of the neutral compound, benzoin, by the−1−1

the region between 1800 cm and 1400 cm . The signal aroundOVM-encapsulated column showed good repeatability in the reten-1657 cm−1 is the amide I band, which mainly consists of υC=O

tion time (RSD=1.23% for the first peak, N=10). Under optimized−1stretching vibrations. The signal at 1549 cm is the amide II band,conditions, the theoretical plate number for the first peak of ben-which mainly consists of υN−H bending vibrations. These two bandszoin reached 72,000 plates/m (Fig. 4).

change its position depending on the structural changes as well as

We prepared a column containing encapsulated trypsin at thethe degree of hydrogen bonding [16]. It is also reported that theinlet of the capillary, and the column digested substrates (an amino

amide I/ amide II intensity and area ratios may be associated withacid derivative and peptides) and separated the substrates and prod-

conformational changes of the protein [16]. The peak (aminde I

ucts within a single capillary (Fig. 5) [17]. We also prepared a col-and amide II) positions of each peak show no differences in both

umn with encapsulated microsomes containing drug-metabolizing

formats. Furthermore, the ratios of peak area are very close withenzymes [18, 19] . This column was used to produce metabolites

each other. These strongly suggest that BSA maintain its confor-and then separate them from the substrates. With this system, themation after encapsulation in the sol-gels [13].

sample size could be reduced by 3 orders of magnitude relative toThe conformational stability of biomolecules is very important to maintain its biological activity. Silica hydrogels contain asufficient amount of trapped interstitial water. Our hydrogel consistof about 80% water in their weight. Therefore the environment
within hydrogel is essentially aqueous, allowing the retention ofFig. 4 Electrochromatogram of benzoin on OVM-encapsulatedcolumn.
Conditions: Sample: 0.1 mg/mL benzoin. Mobile phase: 20 mM
phosphate buffer (pH 7.0)- 2-propanol (95:5, v/v). Applied voltage:
59 V/cm. Injection: 2 kV, 3 s. Column length: 34 cm. Detection:
254 nm.

Fig. 3 IR spectra of BSA in gel or in free solution.

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the same size required for conventional reaction schemes. This system could be also used as an electrophoretic enzyme-inhibitor assay just by injecting an inhibitor onto a column along with thesubstrate-coenzyme mixture [19]. Because the reaction, separation,and detection were integrated into a single system, the system
could be automated, and thus analytical time and the amount ofsolvent required were reduced. Because CE and CEC allow highspeed analysis and efficient separation performance, these results
are very encouraging; and monolithic capillary columns appear toopen new options for the design and construction of highthroughput systems.
Integration of analytical and microfluidic systems has beenused in many fields because the required sample volume is small,
response is fast, highly parallel analyses can be carried out, andFig. 5 The schematic illustration of an on-line enzyme reactor integrated into CE.
A. Substrates are introduced electrokinetically into the enzymeencapsulated reactor.
B. Substrates are catalyzed into products while they flow throughthe enzyme-encapsulated gel by electrophoresis and EOF.
C. The products and unreacted substrates were separated at theseparation section of the capillary by electrophoresis.

cross contamination is minimal [4, 5]. The technique is expected tobe suitable for high-throughput screening. Not surprisingly, sol-gel
technology was used to immobilize biomolecules on microfluidicchips. We used the previously mentioned trypsin-doped hydrogel
for immobilization of trypsin on a microfluidic chip. Large molecules, such as peptides or proteins, were hydrolyzed, and substrate
and product were separated on the microchip (Fig. 6) [20, 21].
4. The immobilization method of membrane proteins using silica nanoparticles
Although alkoxysilanes are widely used for the immobilization of biomolecules, alcohol are produced by hydrolysis of thesealkoxysilanes. These alcohol byproducts will dissolve or destabilize encapsulated proteins or other biomolecules, such as biologicallipid membranes. Therefore, another route using silica nanoparticles and aqueous sodium silicate as a starting material was developed to avoid the production of an alcohol [22]. In this route, silicananoparticles behave as nucleation sites for condensation, andgrow their particle diameter by condensation with sodium silicate.
The grown silica nanoparticles form silica network and trap biomolecules.

Themicrosomes

containing

expressed

drug-metabolizing enzymes, P 450 s, and other electron transfer enzymes were immobilized on the microassay plate using sol-gel
chemistry. A thin-film hydrogel containing microsomes was fabricated using aqueous silicate as a starting material. The transmission
electron microscopy image clearly showed that the nanoclusters derived from the silica nanoparticles formed branched chains, and mi-Fig. 6 Electropherogram of an on-chip tryptic reaction using
NBD-bradykinin as a substrate.
Conditions: Sample: 0.1 mM I. S., 0.18 mM NBD-bradykinin; detection point, 16 mm; I. S. (internal standard), NBDethylenediamine. NBD-bradykinin was digested at the peptide
bond of N-terminal Arg, and NBD−Arg was produced.

crosomes were entrapped in the silica network (Fig. 7). The different P 450 isozymes were immobilized on the microassay plate, andthe metabolites by each isozyme were visualized as fluorescent images, which creates opportunity for the inhibitor assays. As shownin Fig. 8, the immobilized CYP 1 A 1 successfully metabolized
ethoxyresorufin to resorufin, whose fluorescence was visualized. Itwas shown that the fluorescence intensity decreased as the concen-―２０―

CHROMATOGRAPHY, Vol.31 No.1 (2010)

Kumiko Katochemistry and materials science.
Acknowlegement
The author would like to express her gratitude to Professor
Kazuhiro Imai (Professor Emeritus, The University of Tokyo), Professor Toshimasa Toyo’oka (University of Shizuoka), Professor
Hiroshi Homma (Kitasato University), Professor Naoko Utsunomiya-Tate (Musashino University), Associate Professor Tomofumi
Santa (The University of Tokyo), Project Associate Professor
Masaru Kato (The University of Tokyo) for their supervision andvaluable discussions concerning her research. The author greatly
acknowledges all the collaborators and coworkers of her studies.
Financial support from governmental foundation of MinistryFig. 7 Transmission electron microscopy image of microsomes
immobilized in gels fabricated by using silica nanoparticles.

of Education, Culture, Sports, Science and Technology as well asfrom private foundations are gratefully acknowledged.
Finally, the author is grateful to the Society for Chromatographic Science for selecting her as a recipient of the Encouragement Award in 2009 and giving her an opportunity to publish thisfocusing review.
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