An electrospray ionization device and its application

[0048] Example 1 Manufacturing method and structure of electrospray ionization device

[0049] (Production of glass substrate)

[0050] Cut a cover glass with a thickness of 20μm with a glass knife into an isosceles triangle with a waist length of 0.8cm and an apex angle of 45°. After washing and drying, spin a layer on the surface with a homogenizer at 1000rpm The SU-82015 negative photoresist with a thickness of 30μm is baked at 65°C for 20min, and a mask with a microchannel pattern (the width of the microchannel is 2mm, the number is 3) is covered on the glass slide, After ultraviolet exposure, development, and nitrogen drying, a glass substrate with 3 microchannels with a width of 2 mm and a depth of 30 μm is obtained.

[0051] (Production of PDMS chamber)

[0052] The PDMS prepolymer and the initiator are mixed at a mass ratio of 10:1 and then poured on the silicon wafer. The silicon wafer is sealed in advance with a wide tape to prevent the PDMS prepolymer from leaking. After removing bubbles, the PDMS prepolymer was placed at 75° C. for 2 hours to carry out the polymerization reaction. The polymerized PDMS sheet was carefully peeled off, and cut into a PDMS sheet of 2cm×2cm×5mm. Use the same method to make another PDMS sheet of the same size, and dig out an isosceles triangle cavity (the waist length of the isosceles triangle cavity is 1.2 cm, and the vertex angle is 45°). The PDMS sheet with the isosceles triangle cavity dug and the PDMS sheet without the cavity are laminated together to form a PDMS chamber that can accommodate the glass substrate, and the glass substrate is placed in it.

[0053] Conductive clip: prepare a conductive metal clip for use.

[0054] Example 2 Co-cultivation of cells in microchannels

[0055] 1) Digest the human liver tumor cells HepG2, human cloned colon adenocarcinoma cell Caco-2 and human breast cancer cell MCF-7 cells that are overgrown in a 60mm petri dish, and dilute them with culture medium to 10 6 mL -1 Cell suspension. Take 10 μL of the cell suspension of the three kinds of cells and add them to the three microchannels on the glass substrate in the PDMS chamber (e.g. figure 2 (Shown), after the cells adhere to the wall, add some new medium, put the glass substrate placed in the PDMS chamber into the cell incubator, and culture overnight.

[0056] 2) Take out the glass substrate from the incubator, stain HepG2, Caco-2 and MCF-7 cells with three different fluorescent dyes, Cell TrackerTM Green CMFDA, CellTrackerTM Deep Red and Cell TrackerTM Violet, and place them in a confocal laser Observe under the microscope, the results are as image 3 Shown. It can be seen that due to the surface tension of the microchannel, the three kinds of cells grow in their respective microchannels, and the cells will not mix with each other.

[0057] 3) In the same way as in step 1), culture the three types of HepG2, Caco-2 and MCF-7 cells overnight, and change the medium every day for standby. The cell survival rate was tested daily with a deadly kit (Calcein-AM/EthD-1). The result is Figure 4 As shown, the cell activity remained normal during the first three days of culture, and the cell survival rate still reached over 85% on the third day.

[0058] Example 3 Qualitative mass spectrometry detection of drug absorption in cells

[0059] Co-culture HepG2, Caco-2 and MCF-7 cells in the same manner as step 1) of Example 2. After culturing overnight, remove the glass substrate placed in the PDMS chamber from the incubator, and add cyclophosphamide dropwise The solution (hereinafter referred to as CPA, the concentration is 10 mmol/L, that is, 10 mM, the dosage is 10 μL), and then put it back into the incubator for 24 hours.

[0060] A glass substrate with MCF-7 cells (ie, monocultured MCF-7 cells) cultured in all three microchannels was used as a control sample, and the culture mode and drug action mode were the same as the co-cultured MCF-7 cells.

[0061] Remove the glass substrate placed in the PDMS chamber from the cell incubator, clamp the glass substrate, and rinse with distilled water three times to remove the complex matrix remaining on the surface of the glass slide. After the glass substrate is naturally dried, place it in front of the Shimadzu LCMS-2010A mass spectrometer with a conductive clip, so that the tip of the glass substrate (at an angle of 45°) faces the inlet of the mass spectrometer, and apply the conductive clip to the glass substrate High voltage (~4.5kV), while adding isopropanol as an organic solvent into the microchannel where the MCF-7 cells are located, an electrospray is generated immediately, and the spray ions are collected by a mass spectrometer and scanned and analyzed. The mass-to-charge ratio (m/z) of the scan ranges from 100 to 500, and the detection is performed in the positive ion mode. The control sample was also scanned and analyzed in the above-mentioned manner.

[0062] Such as Figure 5 with 6 As shown, there is [CPA+H] in all mass spectra + Peak (m/z is 262), [CPA+Na] + Peak (m/z is 284) and [CPA+K] + Peak (m/z is 300). These typical mass spectrum peaks indicate that CPA is absorbed in both monoculture and co-cultured MCF-7 cells.