A NEW DISCRIMINATION METHOD OF TARGET BIOMOLECULES
WITH MINIATURIZED SENSOR ARRAY UTILIZING LIPOSOME
ENCAPSULATING FLUORESCENT MOLECULES WITH TIME COURSE
ANALYSIS
K. Takada1, T. Fujimoto1, T. Shimanouchi2, M. Fukuzawa1, K. Yamashita1, H. Umakoshi3
and M. Noda1*
1
2
Kyoto Institute of Technology, JAPAN, Okayama University, JAPAN, and 3Osaka University, JAPAN
ABSTRACT
This paper reports a new type of detection approach by arrayed biosensor for discrimination of target biomolecules
such as protein and/or evaluation of their concentrations, utilizing liposome encapsulating fluorescent molecules and the
corresponding time course analysis of the fluorescence. This method is realized by obtaining multi-dimensional data plots
(curve) of fluorescent output such as its intensity from plural and different liposomes independent from each other. Two
different proteins (carbonic anhydrase from bovine: CAB and Lysozyme), including their concentrations, were clearly
discriminated by the developed method.
KEYWORDS: Discrimination method, Sensor array, Liposome, Target biomolecule, Protein, Fluorescence
INTRODUCTION
The approach to study the protein-lipid membrane interaction has been developed to clarify the membrane–related
phenomena in cell system, such as the endocytosis, the exocytosis and the membrane fusion. A systematic investigation
on the membrane properties as a function of the lipid composition is expected to contribute to the understanding not only
on the phenomena in cell system but also on the effective design of novel biomaterials. The information as a whole in relation to the lipid composition, the membrane property, the phase state of lipid membrane, or the function induced on lipid (liposome) membrane could be called as “Membranome [1]” according to the previous manner of genome or proteome. In the Membranomics research, the adequate methodology is needed to evaluate the interaction of lipid (liposome)
membrane with the biomaterials such as peptides/proteins or liposomes. In a series of reports, the immobilization technique of intact liposome on solid surfaces has been developed to apply to the investigation of the protein–liposome interaction, and the membrane-membrane interaction. In the study in relation to the protein–liposome interaction, the systematic information for the surface property of proteins could be acquired. Thus, a Membranome information of lipid
(liposome) might be elucidated from a systematic investigation on the protein–liposome interaction and membrane–
membrane interaction.
A part of the authors have examined a membrane chip analysis of fluorescence detection for liposome-liposome interaction [2,3] but not for liposome-protein interaction, and not by a miniaturized chip system. In this study, we tried the detection of protein as target biomolecule by using a developed miniaturized chip system.
EXPERIMENTAL
Key points in this work are as follows; 1) 3 x 3 arrayed 700 m square microwell structure was fabricated for
immobilizing droplet (0.3 L) of liposome suspension with solution of target protein (0.3 L), 2) three different
liposomes of 1,2-distearyl-phosphatidylcholine(DSPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine(POPC) and 1,2dipalmitoyl-phosphatidylcholine(DPPC) were selected as biosensing molecules because of their difference in fluidity of
lipid membrane at an ambient temperature when interacting with target proteins, 3) a compact fluorescence detection
system was developed especially for obtaining information from fluorescence output such as intensity, its distribution,
wavelength and its distribution in every independent pixel in the array, respectively. The main principle of detection and
discrimination of target protein is based on the difference in interaction behavior, basically hydrophobic interaction [2,3],
between the individual lipid membrane and target protein. To our knowledge, there seems to be little reports on 2-D
liposome arrays analyzing characteristics of leaked fluorescent molecules encapsulated in the liposome by interaction
with target protein, although a number of liposome arrays with different detection principles have been reported[e.g. 4,5].
RESULTS AND DISCUSSION
Figure 1 illustrates a cross-sectional view of microwell chambers of fabricated sensor array and examples of its
fluorescence image. A droplet of suspension (30 mM) of liposome (DSPC, POPC, and DPPC, in order) encapsulating
calcein solution (100 mM) was spotted in a microwell by a semi-automatic microfluid dispenser, thereafter a solution of
target protein (Lysozyme, CAB, and ultrapure water as control, in order) was supplied. The top of the microwell was
covered by a glass plate to avoid vaporization of solvent. In a fabricated photometric system (Fig. 2), the calcein
excitation light (around 495m in wavelength) is emitted from a blue-LED and calcein fluorescence through an optical
filter (502-730 nm) was detected by a CCD imager. The obtained fluorescent images for every pixel in the array were
analyzed. Figure 3 shows a time course of increase in fluorescence (green: around 546 nm) leaked from DPPC liposome.
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17th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
27-31 October 2013, Freiburg, Germany
We find that the interaction behaviors are definitely different between the three targets. Figure 4 shows a 2-D correlation
chart of fluorescence intensities from liposomes of DSPC and DPPC. It was observed from the plot that the two different
proteins were sufficiently discriminated with their concentrations. This method would allow us to improve the precision
of analysis by combining with statistical processing such as principle component analysis and/or multiple regression
analysis.
Figure 1: A cross-sectional view of microwell chambers of fabricated sensor array and examples of its fluorescence
image
Figure 2: An illustration of photometric system for measuring fluorescence signal from the liposome array chip
Figure 3: Time course of increase in fluorescence for lysozyme(710 M), CAB(350M) and ultrapure water leaked
from DPPC liposome (n=3)
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Increased ratio of fluorescence
in DSPC liposome (%)
CAB 350M
CAB
Lysozyme
CAB
200M
Lysozyme
300M
Water
CAB 35M
Lysozyme 710M
Lysozyme
71M
Increased ratio of fluorescence
in DPPC liposome (%)
Figure 4: A 2-D correlation chart of fluorescence intensities from liposomes of DSPC and DPPC (n=3)
CONCLUSION
A new type of detection approach by arrayed biosensor was developed for discrimination of target biomolecules such
as protein and/or evaluation of their concentrations, utilizing liposome encapsulating fluorescent molecules and the corresponding time course analysis of the fluorescence. By obtaining multi-dimensional data plots (curve) of fluorescent output
such as its intensity from plural and different liposomes independent from each other, two different proteins (carbonic anhydrase from bovine: CAB and Lysozyme), including their concentrations, were clearly discriminated by the developed
method. It is revealed that the interaction behaviors between the liposomes and target protein are definitely different between the two protein targets. Furthermore, from the 2-D correlation chart of fluorescence intensities from the liposomes,
it was observed that the two different proteins were definitely discriminated with their concentrations.
ACKNOWLEDGEMENTS
This research was supported in part by Grants-in-Aid for Scientific Research (No. 19360162, 21246121, 20760539)
from the Japan Society for the Promotion of Science.
REFERENCES
[1] R. Kuboi, H. Umakoshi, T. Shimanouchi, “Cutting Edge of Membrane Stress Biotechnology”, MEMBRANE, vol.
33(6), pp.300-306, 2008.
[2] T. Shimanouchi, E. Oyama, H. Ishii, H. Umakoshi, R. Kuboi, “Membranomics Research on Interactions between
Liposome Membranes with Membrane Chip Analysis”, MEMBRANE, vol. 34, pp.1-9, 2009.
[3] T. Shimanouchi, E. Oyama, H. Thi Vu, H. Ishii, H. Umakoshi, R. Kuboi, “Monitoring of membrane damages by dialysis treatment: Study with membrane chip analysis”, Desalination and Water Treatment, vol. 17, pp. 45-51, 2010.
[4] M. Bally, K. Bailey, K. Sugihara, D. Grieshaber, J. Vörös, B. Städler, “Review- Liposome and Lipid Bilayer Arrays
Towards Biosensing Applications”, Small, vol. 6 (22), pp.2481–2497, 2010.
[5] B. Chaize, M. Nguyen, T. Ruysschaert, V. le Berre, E.Tre´visiol, A-M. Caminade, J. P. Majoral, G. Pratviel, B.
Meunier, M. Winterhalter, and D. Fournier, “Microstructured Liposome Array”, Bioconjugate Chem., vol. 17 (1),
pp.245–247, 2006.
CONTACT
*Minoru Noda, tel: +81-75-724-7443; [email protected]
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