Introduction 1 2 3 8 9 11 12 13 14 15 16 17 18 19 20 21 22 In this paper, we report preliminary results concerning the preparation of a CAs-CHIP as a deproteinization attachment for CE separation. Deproteinization was achieved by use of the multilayer flow obtained in the PDMS microchannel, and the small molecules separated from the mixed protein sample were injected into the separation capillary connected directly to the CAs-CHIP, to be analyzed by CE. In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species, and feasibility study was performed. Experimental Square capillaries and reagents 7 −1 Fabrication of CAs-CHIP-CE system by embedding square capillaries on a PDMS plate and bonding of a PDMS cover 1 1 1 18 18 2 Fig. 1 General concept for fabricating a diffusion-based pretreatment–CE separation system using a capillary-assembled microchip (CAs-CHIP). The plugged capillaries indicated as gray parts are actually square capillaries with 50 μm square-shaped conduits blocked with PDMS. In this figure, for simplicity, these conduits are not shown  Fig. 2 Photograph of a fabricated CAs-CHIP-CE device  Operating procedures −1 −1 −1 −1 −1 1 Capturing fluorescence images and laser-induced fluorescence measurement Fluorescence images of the microchannel were obtained by using an optical/fluorescence inverted microscope (Eclipse TS100-F, Nikon, Tokyo, Japan). Photographs were captured using a 3CCD color camera (HV-D28S, Hitachi Kokusai Electric, Tokyo, Japan) installed at the front port of the microscope. Fluorescent images were collected using a mercury lamp as a light source and a filter block (G-2A and FITC, Nikon, Tokyo, Japan). CE with laser-induced fluorescence (LIF) detection was performed on a home-built system based on an inverted fluorescence microscope (IX70, Olympus, Tokyo, Japan). Light at 488 nm from an argon ion laser (Newport Spectra Physics Laser Division, Mountain View, CA, USA) was introduced into the microscope. The laser beam was filtered through a 460–490 nm band-pass filter, reflected by a 510 nm dichroic mirror, then focused on the detection point by means of a 20× objective lens. Fluorescence was collected by use of the same objective lens, filtered through a 515 nm high-pass filter, and finally detected by use of a CCD camera (Model PMA-11, Hamamatsu Photonics, Shizuoka, Japan). To obtain electropherograms fluorescence at 600 nm was used throughout. Results and discussion Optimization of diffusion-based deproteinization 23 23 24 3 3 3 3 y Fig. 3 a −1 −1 −1 b −1 −1 −1 b  25 −6 2 −1 −7 2 −1 26 27 Sample injection and electrophoretic separation 1 28 n 4 −1 −1 −1 −1 Fig. 4 −1 −1  Deproteinization and electrophoretic separation 5 5 29 5 5 5 R R Fig. 5 −1 −1 −1 −1  Conclusions We have demonstrated deproteinization and CE separation on a single device using CAs-CHIP technology. Diffusion-based deproteinization in a CAs-CHIP was successfully achieved by choosing an appropriate flow rate, and subsequent CE separation of small molecules was achieved by use of a separation capillary connected directly to the CAs-CHIP. Because diffusion-based separation is, in general, based on dilution of the sample solution, preconcentration before CE separation will be required in the next step. Because the CAs-CHIP is prepared by simply embedding square capillaries, however, further integration of the preconcentration process using chemically-functionalized capillaries, or the on-line preconcentration techniques reported to date, can be also used. These applications are currently under investigation.