Detection device and detection method of dot immunoblotting detection

A detection device of dot immunoblotting detection, comprising:

• a negative pressure suction device;
• a hole plate;
• a hose for connecting the hole plate with the negative pressure suction device;
• and
• a nitrocellulose membrane in tight fit with an upper end surface of the hole plate;
• wherein the hole plate comprises an upper-layer supporting plate, a lower-layer tray hermetically connected under the upper-layer supporting plate, and a negative pressure cavity arranged between the upper-layer supporting plate and the lower-layer tray;
• wherein one side of the lower-layer tray is provided with a connection pipe for allowing the hose to be in sleeve connection, and a plurality of through holes are formed in the upper-layer supporting plate;
• and
• wherein a size of the nitrocellulose membrane is the same as a size of the upper end surface of the hole plate.

A detection method of dot immunoblotting detection, comprising:

• connecting a portable sputum aspirator with a hole plate through a hose, cutting a nitrocellulose membrane into a size which is the same as the size of an upper-layer supporting plate on the upper end surface of the hole plate, placing the nitrocellulose membrane onto the hole plate having a plurality of through holes, turning on the portable sputum aspirator, setting a negative pressure value of the portable sputum aspirator to be approximately 0.06 MPa, and forming a plurality of sample injection grooves in the surface of the nitrocellulose membrane;
• dividing the plurality of sample injection grooves into reference substance holes and sample holes, each repeated for three times, separately adding a reference substance and approximately 1 μL of a sample to be tested into each reference substance hole and each sample hole, and naturally air-drying the nitrocellulose membrane for approximately 30 minutes at the end of the sample injection;
• placing the nitrocellulose membrane into an approximately 5% skim milk powder solution, placing the skim milk powder solution onto a shaking table for blocking at room temperature for approximately 1.5 hours, abandoning the blocking liquid, and rinsing the membrane with TBS-T, wherein the skim milk powder solution is diluted with a TBS-T solution until the concentration is approximately 5%;
• diluting a primary antibody with an approximately 5% bovine serum albumin solution in a ratio of approximately 1:500, uniformly and completely immersing the nitrocellulose membrane into the diluted primary antibody, placing the mixture onto the shaking table, carrying out constant-temperature incubation at approximately 37° Celsius for approximately 4 hours, and placing the nitrocellulose membrane into the TBS-T at the end of the incubation for washing on the shaking table, for approximately 5 minutes at each time, wherein the bovine serum albumin solution is diluted with the TBS-T solution until the concentration is approximately 5%;
• diluting a secondary antibody with an approximately 1% skim milk powder solution in a ratio of approximately 1:5000, uniformly and completely immersing the nitrocellulose membrane into the diluted secondary antibody, placing the mixture onto the shaking table, carrying out incubation at room temperature for approximately 1.5 hours, and placing the nitrocellulose membrane into the TBS-T at the end of the incubation for washing, for approximately 15 minutes at each time, wherein the 1% skim milk powder solution is diluted with the TBS-T solution until the concentration is approximately 1%;
• laying the washed nitrocellulose membrane at a proper position of a developing device, uniformly adding dropwise a developer onto the nitrocellulose membrane, and using a gel imaging system to carry out exposure and development, and saving an image;
• and
• scanning and analyzing, by means of Image J software, a gray value of an image obtained by the development, and expressing a relative concentration of a target protein of each sample by using a gray value of a reference sample.