An open-space microfluidic chip with fluid walls for online detection of VEGF via rolling circle amplification† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc02974e

We report an open-space microfluidic chip with fluid walls, integrating functions of cell culture and online detection of secreted proteins controlled by the interfacial tension value.


Table of Contents:
Materials and methods S-2

Materials and methods
Reagents.

Fabrication of Microfluidic Devices.
A home-made PDMS-based microfluidic chip, consisting of two major round chambers and a narrow connecting channel, was pre-made according to the conventional procedures 1 . The PDMS chip could be used repeatedly in all following experiments, reducing the process of remodeling. A confocal dish with its inside side of the bottom covering with the PDMS chip was processed with oxygen plasma. The regions without coverage of the PDMS chip would be treated by plasma and became hydrophilic. After sterilization with ultraviolet for 30 minutes, the cell medium or water solution for modification could be injected to the round chambers, and then the narrow channel could be connected if necessary controlled by interfacial surface tension. In order to prevent evaporation of aqueous solution, oil phase was overlaid on the bottom of the glass. Now the open-space microfluid chip with fluid walls was completed.
Cell Culture and Staining.
All cells were cultured in minimum essential medium (MEM) with extra supplement of 10% FBS and 1% penicillin−gentamicin at 37 °C in a humidified incubator containing 5% CO 2 by volume. To ensure sufficient nutrition for cell normal growth, the cells were trypsined and reseeded every two or three days. Both the cell subculture and pretreatment were conducted at a clean bench. Before the experiment, cells were dealt with trypsin-EDTA and resuspended with 1 mL MEM medium. The cell suspension was reinjected into the cell S-2 culture chambers until absolute adhesion on the glass. In addition, in order to ensure satisfactory cell viability in such a platform, cells were stained with Calcein-AM and PI to indicate live or death.

Modification of the Microfluidic Devices
Turn "OFF" the connection between the two functional chambers and modify the surface on the signal detection chamber. First, the hydrophilic part of the chamber was treated with the H 2 SO 4 /H 2 O 2 solution (v/v,3:1) to keep the surface with hydroxy and then washed with purified water for three times. Second, carboxyl group was modified on the bottom of the chamber processed with carboxylated silancance (TCS) for 2 h. After that, the chamber was washed with PBS buffer (pH=7.4) three times. Next, a mixture of NHS solution (0.4 M) and EDC solution (1.6 M) at the ratio of 1:1 (v:v) was introduced and incubated for 30 min at room temperature.
Finally, the first DNA aptamer for VEGF (4 μM) was injected to the chamber. After incubating in 37 °C for 30 min, the aptamer solution was removed by PBS washing. Now the bottom of microfluidic device was modified with DNA aptamers and are ready for the capture of VEGF protein molecules.

Evaluation of rolling circle amplification.
A pre-mixed RCA reaction system was detected by fluorescence spectrum to verify the efficiency of RCA reaction. The second circle template aptamer was linked to padlock DNA sequence via T4 DNA ligase in advance, and the mixture of dNTP, phi 29 DNA polymerase promoted the RCA reaction process. In addition, a negative group without dNTP and phi 29 DNA polymerase was detected simultaneously. SYBR gold could combine with DNA fragment after amplification and the fluorescence signal could be excited with 495 nm laser, collected in 540 nm wavelength.

Rolling Circle Amplification (RCA) Reaction and VEGF Detection.
After samples containing VEGF added into the signal detection chamber, the device was incubated at 37 ℃ for 30 min to immobilize VEGF adequately. Then the chamber was blocked with 10% FBS in PBS buffer and washed with PBS for three times. After that, a premixed solution of the second DNA aptamer ligation template (10 μM, 2 μL), padlock DNA (10 μM, 2.5 μL), T 4 DNA ligase (400 U/μL, 1 μL) and 10× ligase buffer (1.5 μL) was injected to the chamber at 37 C for 30 min. After washing with PBS for three times, the RCA reaction took place for 30 min at 37 °C in a mixed solution with 1 μL phi29 (1 U/μL) polymerase, 2 μL dNTP (2.5 mM), 0.5 μL BSA (10 mg/mL), 2 μL phi29 buffer (50 mM Tris·HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , pH=7.5). After the specific binding, the modified chamber was washed with PBS for three times. Finally, the fluorescence DNA probe was added into the chamber, incubated for 10 min at room temperature and then washed with PBS three times for fluorescence imaging.

Imaging and counting.
After completing the processes above, the confocal dish was taken to image using FV 5000 Spinning disk.