Self-organized functional lipid vesicle array for sensitive immunoassay chip

ARTICLE IN PRESS Ultramicroscopy 108 (2008) 1325– 1327

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Self-organized functional lipid vesicle array for sensitive immunoassay chip Hea Yeon Lee a,, Bong Kuk Lee b, Jong Wan Park a, Ho Sup Jung a, Jong Min Kim c, Tomoji Kawai a, a b c

The Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Honcho, Kawanguchi, 332-0012 Saitama, Japan Department of Chemical Engineering, Dong-A University, 840 Hadan-dong, Saha-gu, Pusan 604-714, Republic of Korea

a r t i c l e in fo

PACS: 68.37.Ps 82.80.Fk 82.47.Rs Keywords: Biomolecule array Biochips Biosensor Functional lipid vesicles Nanometrics Electrostatic interaction

abstract We report the self-assembly immobilization of functional lipid vesicles (FLVs) by electrostatic interaction onto N-inscription-nanosized geometrics. The well-organized three-dimensional physical structures of liposome were observed by AFM. Generally, two involved forces for the binding to surfaces and the repulsion between individual liposome are necessary to array lipid vesicles individually similar to the physical configuration in solution. The immobilized FLVs demonstrated clearly defined redox activity in electrochemical measurements. We observed a notable current decrease, indicating the binding of the capture antibody with the target human serum albumin (HSA) antigen. We believe these findings can be related to various vesicles applications such as drug delivery system, nanobiosensors and nano-scale membrane function studies. & 2008 Elsevier B.V. All rights reserved.

1. Introduction Advanced biosensor chip has attracted a great deal of attention due to the ever increasing numbers of biological analysts in the clinical, environmental, and bioindustrial fields. The key issues of advanced biochip protocol are the fabrication of appropriately designed nanostructure, the nanoarray retaining bioactivity, and the high-specific biomolecule recognition. More recently, we previously reported the nanometrics geometry of a well-oriented nanowell array by nanolithography method [1]. In the nanosized metrics, most of the area of the metal electrode was covered with the blocking layer, and only a nano-scale metal surface becomes exposed to the open space above the nanowell. The depth and width of the nanosized well can be adjusted to allow for only one or a few biomolecules to enter inside the nanowell and become immobilized with the self-assembled-modified metal surface (Fig. 1(a) and (b)). Meanwhile, functional lipid vesicles (FLVs) membranes have been exploited for various applications such as fundamental biological research of cell-membrane, lipid-based bioassay and biosensor [2–7]. An understanding of FLVs membrane structure and the function of molecules within the membrane is pertinent to issues such as drug delivery across a membrane and numerous cellular regulatory processes. In particular, the lipid vesicle

 Corresponding authors. Tel.: +81 6 6879 8447; fax: +81 6 6875 2440.

E-mail addresses: [email protected] (H.Y. Lee), [email protected] (T. Kawai). 0304-3991/$ - see front matter & 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ultramic.2008.04.089

nanoarray has been used to study cell behavior on modified solid surfaces, and to fabricate lipid-assisted biochips such as DNA chips, protein chips, cell chips, neuron chips [2,7–11]. Various methods have been developed for the fabrication of lipid vesicles arrays such as electron beam lithography [12], soft lithography [13,14], photolithography [15], polymer lift-off [16], modification of surface by UV exposure [17], and scanning probe lithography [18]. Most of these methods utilized physical or chemical modification to the surface for restricting the diffusion of lipids, since the fluidity is an intrinsic nature of vesicles in cell membrane [19,20]. Investigation of the dynamics and structures of liposomes depend on various optical techniques such as a light scattering method, a spectroscopic method and a fluorescence microscopy method. Currently, the best magnified observation comes from high-resolution surface microscopy techniques though immobilization procedures are still required. Thereby, if the FLVs structures similar to those in liquids can be maintained in the solid surface, the critical problem concerned with the biomolecule immobilization will not prohibit the analysis of biomolecule interaction. Meanwhile, the nature of the inside water pool promises the usability of FLVs to the various functional systems. Threedimensional FLVs structures similar to those in the liquid phase have to be sustained in the surfaces to investigate the dynamics related to the inside water pool. The physical instabilities of FLVs render strong mixing difficult and engender the different concentration distribution on the local surface. Individual liposome, however, easily aggregates with each other, ultimately forming giant vesicles and aggregators on hydrophobic surface.

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repulsion

Fig. 1. Picture of the nanowell-arrayed electrode, which the size of the whole substrate is 2.5  2.5 cm2 (a), schematic illustration of biomolecule interaction onto nanowell array electrode (b) and of effective immobilization of FLVs onto e-beam exposed surface (c).

In this paper, we present the self-assembly FLVs nanoarray, while preserving their stability, onto nanogeometric gold electrode by controlling electrostatic interaction as shown in Fig. 1(c), and the electrochemical immunosensor chip for detection of the model proteins human serum albumin (HSA) and carbonic anhydrase from bovine (CAB) using protein G attached streptavidin on modified FLVs micro-array electrode. The lipid vesicles nanoarray is a prerequisite for the development of highthroughput biosensors and for performing chip-based studies of cellular interactions based on self-assembly of lipid bilayers.

2. Experiment 2.1. FLVs preparation The FLVs were prepared by an extrusion method [21]. To obtain FLVs, a lipid solution was prepared by mixing 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3phospho-1-glycerol (DMPG), 1,2-dioleoyl-phosphatidylethanolamine-N-caproyl-amine (Cap-PE), N-(10,12-Pentacosadiynoic) acetylferrocene (Fc-PDA), cholesterol, and 1-octadecanthiol with a molar ratio of 10:10:1:1:1:0.1. All samples were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). The Fc-PDA will be used as a facile redox probe for electrochemical detections. The lyophilized solution was rehydrated using a phosphate-buffered solution (PBS), and then treated with five freeze-thaw cycles. The lipid vesicles were extruded repeatedly through a polycarbonate film with 100 nm pores using an extrusion device (LipoFast; Avestin Inc.) to produce a liposome solution. Final concentration of the phospholipids was approximately 10 mM. The lipid vesicle size was confirmed by the dynamic light scattering method (DLS-700 Ar; Otsuka Electronics Co., Ltd., Japan). 2.2. Preparation of nanogeometric electrode and immobilization of FLVs An e-beam lithography technique was used for the fabrication of an immobilization substrate using a standard positive polymer resist (ZEP520; Zeon Corporation). Before the e-beam process, the gold layer of 200 nm has been fabricated on the Ti sub-layer (10 nm) by a sputtering technique. Next, the resist was spincoated on the whole substrate with a thickness of 300 and 150 nm

before exposed to the e-beam. The used dose was about 150 C/cm2 using a 75 kV scanning electron microscope (ELC-2; Elionix Co. Ltd.). The immobilization of the liposome was performed by incubating a 10 ml portion of the liposome solution on the e-beam-exposed substrate for 1 h. The substrate was washed with buffer solution. The FLV-modified electrode was then carefully rinsed with 100 mM PBS solution to remove any unbound FLVs. To immobilize the capture antibody (anti-HSA), 2 ı´L of protein G-conjugated streptavidin (10 mg/mL) was imposed upon the FLV-modified electrode for at least 30 min and was then washed with Millipore Milli-Q (18 MO cm) water. A 10 mg/mL portion of biotinylated anti-HSA was dropped on the modified electrode for 30 min, and target proteins (10 mg/mL) were injected successively for 15 min at 37 1C. All surface treatment processes were carried out at room temperature in a high humidity environment. 2.3. Measurement A conventional AFM instrument (Dimension 3100, Digital Instruments, USA) was used for surface imaging. All measurements were performed using the intermittent contact mode (tapping mode) with a scanning speed of 1 Hz (512  512 data format) in the air. Tapping mode tips with spring constant 0.9–2.2 mN were obtained from OLYMPUS (Japan). The FLV solution was injected into the flow cells with 100 mM PBS (pH 7.4) as carrier buffer solution. Protein G-conjugated streptavidin, anti-HSA, 0.1 mg/mL BSA (blocking solution), and HSA were successively injected at a flow rate (5 mL/min). Electrochemical measurements were performed on a BAS100 B/W potentiostat (Bioanalytical Systems, Inc.) at room temperature. Electrodes used were gold working electrodes, an Ag/AgCl reference electrode in 3 M KCl, and a platinum-wire counterelectrode (1 mm). Squarewave voltammetry (SWV) measurements were performed in a solution containing 100 mM PBS solution (pH 7.4) at a scan rate of 100 mV/s. SWV was performed with the following parameters: 0.2 V initial potential, 0.8 V end potential, 25 mV amplitude, 4 mV step potentials, and 50 Hz frequency.

3. Results and discussion Fig. 2(a) and (b) shows AFM topographic images of N-inscription-nanosized geometrics before and after immobilization of 100 nm FLV, which show 200 nm-sized holes developed by the e-beam lithography. The examined thickness of the resist ranged from about 200 nm. It was offered the confirmation that the highly dense immobilization of liposome is located inside N-well electrode. The three-dimensional physical structure is not perfectly ideal but a sufficiently well-organized shape than various previous reports. Although the fabricated liposome has both neutral and anionic lipids, the local charge distribution of the liposome can be rearranged by a rafting process. The rafted anionic charge leads to repulsion between the each individual liposome and the electron accumulated resist surface. It was reported that various e-beam-exposed non-conducting polymers have wide ranges of the electron accumulation [22]. Thereby it was accumulated by electrons in the resist during the e-beam lithography process. Therefore, the immobilization of FLVs inside nanometrics is driven by an electrostatic interaction between the surface and the liposomes as illustrated in Fig. 2(b). Fig. 3(a) illustrates the principles of direct antibody–antigen interaction on FLV-modified microelectrode as immunosensor. Protein G-modified streptavidin, which was directly attached FLVs on substrate and is used to immobilize the capture antibody, plays

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signals of the FLV electrode decreased noticeably when the HSA interacted with the FLV sensors. This result suggests that the target antigen binding to anti-HSA on the FLV’s membrane subsequently blocks the electron transport path into the electrode. Therefore, the stacking of target antigen onto the FLV leads to the suppression of further electron-transfer currents. The results suggest that biotin–streptavidin interaction may be useful for immobilization of the antibody and nonspecific binding processes could be avoided and background interaction reduced to a minimum by immobilization of FLVs.

4. Conclusion

Fig. 2. AFM topographic images of N-inscription-nanosized geometrics before (a) and after (b), immobilization of FLVs which show 200-nm-sized wells.

We reported the self-organized FLVs array on nanometrics electrode using electrostatic interaction. The immobilized FLVs maintain their three-dimensional configuration similar to those in the liquid. The main factor for the successful immobilization is a strong static charge interaction but more careful investigation for the surface chemistry is still required in the future. We believe that the immobilization method is useful not only for surfacerelated liposome observation, but also for many liposome-related applications by designing the shape and the structure of various nanolithography. In addition, we have confirmed the electrochemical activity on liposome-modified solid surface. The capture antibody can be immobilized firmly and apparently made to interact specifically with the target antigen, which retains its orientation and bioactivity. It is hoped that this simple method provides an alternative platform for the fabrication of lipid-based immunoassay chips and a useful tool for research of lipid membrane within microfludic devices.

Acknowledgment

Fig. 3. Schematic illustration of liposome-based immunosensor system (a), SWVs obtained upon detection of target HSA protein (b) and negative target protein (c) on protein G-streptavidin-modified liposome electrode. Inset: cyclic voltammograms of the liposome layer in both the presence and absence of Fc-PDA probes in membranes.

a key role in the binding process between the receptor and the FLV’s sensor ligand. The biotinylated capture antibody (anti-HSA) was firmly immobilized as a receptor onto the FLV membrane by streptavidin–biotin interaction. The FLVs themselves are composed on supramolecular assemblies containing Cap-PE, cholesterol, and a facile redox probe (Fc-PDA), which is important for electrochemical detection. Finally, the antigen was captured by the antibody and then the immunosensor was evaluated by observation and measurement of the current change before and after injection of the antigens. The electrochemical measurement of the liposome layer in the presence and absence of Fc-PDA liposome membrane probes in 100 mM PBS solution is as illustrated in the inset of Fig. 3(a). Well-defined current responses were obtained for the Fc-PDA-containing liposome electrode. However, no redox response was observed for the immobilized liposome in the absence of Fc-PDA. For that reason, it is supposed that the liposome modified with Fc-PDA can be used to detect the target antigen by redox current changes on the immobilized electrode. Fig. 3(b) shows SWV results obtained immediately after the immobilization of anti-HSA and interaction of HSA on the FLVs. Current responses of the FLV electrodes decreased successively following immobilization of anti-HSA and HSA. Current

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