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How to use microchip

Protocol of microchips using for clinical laboratory diagnostics

1. Obtain blood serum using standard protocol

2. Put microchips and sera to a robotic hybridization station

3. After finishing of hybridization procedure measure fluorescence intensity using a fluorescent reader for microchips

4. Analyze your data using the software "Semiotik" for PC (release in 2016).


Manual protocol of microchips development for scientific research

Microchip development without robotic hybridization station

1. Obtain blood serum using standard protocol;

2. Keep the microchips for 15 min in 0.1 M isotonic phosphate buffer, containing 0.1% of Tween 20 (0,1% PBS).;

3. Dilute blood serum at 1 to 15 in appropriate volume of 1% phosphate buffer saline, pH 7.4 (PBS), containing 3% BSA and incubate for 10 min at 37°C;

4. Centrifuge a sample for 15 min at 12 000 rpm;

5. Aspirate an upper part of sample and apply it onto microchip surface;

6. Keep microchip on a shaker for 1.5 h at 37°C and 80% of relative humidity

7. Wash the chips 0.05% PSB and add a solution of biotinylated goat antibodies recognizing human IgG, M and A in appropriate dilution in 0.1% PBS or a solution containing goat antibodies recognizing human IgG labeled with Alexa555 (or Cy3, or analogous) and human IgM labeled with Alexa647 (or Cy5, or analogous) - in appropriate dilution in 0.1% PBS;

8. Incubate microchip on shaker for 1 h at 37°C and 80% of relative humidity (in the dark in case of fluorescently labeled antibodies) and wash the chips with 0.05% PBS;

9. In case of biotinylated secondary antibodies add a solution of fluorescence labeled (Alexa 555 (or Cy3) or Alexa 647 (or Cy5) streptavidin - in appropriate dilution in 0.1% PBS and keep for 0.5 h at room temperature in the dark;

10. Wash the chips with 0.05% PBS followed by MilliQ (or bidistillated water);

11. Dry microchips with centrifugation or in nitrogen stream;

12. Measure the intensity of fluorescence using a fluorescent reader for microchips at 5 - 10 μm resolution;

13. Convert obtained images to Excel using reader's software and GAL.-file (supplied with chips);

13. Analyze your data using the software "Semiotik" for PC (available for registered users).


Microchip development using robotic hybridization station

1. Obtain blood serum using standard protocol;

2. Put microchips and sera to robotic hybridization station;

3. After finishing of hybridization procedure measure a fluorescence intensity using fluorescence intensity using fluorescent rider for microchips;

4. Convert obtained images to Excel using reader's software and GAL.-file (supplied with chips)

5. Analyze your data using the software "Semiotik" for PC (available for registered users).


Buffer preparation

Preparation of isotonic phosphate buffer

Weigh 8.00 g of sodium chloride, 0.27 g of potassium dihydrophosphate, 0.20 g of potassium chloride, 3.58 g of sodium hydrophosphate heptahydrate using laboratory balance, transfer to a 1 000 mL bottle and add 900-950 mL of MilliQ or bidistilled water. After dissolution of reagents, bring solution to the volume with water. If necessary, adjust pH value to 7.4. It is necessary to pass the buffer through a 0.22 μm filter before each manipulation. Concentrations of buffer components are provided in Table 1.

   Component          Concentration     
      KH2PO4           2 mM
     Na2HPO4          10 mM
        NaCl        137 mM
         KCl         2.7 mM



The obtained solution is stable for one week when  stored at +4°C.

Preparation of isotonic phosphate buffer containing 1% of Tween 20

Add 10 mL of Tween 20 to 1 I of filtered isotonic phosphate buffer. Mix till complete dissolution. The obtained solution is stable for one week when stored at 4°C.

Preparation of isotonic phosphate buffer containing 0.1% of Tween 20

Add 1.0 mL of Tween 20 to 1 I of filtered isotonic phosphate buffer. Mix till complete dissolution. The obtained solution is stable for one week when stored at 4°C.

Preparation of isotonic phosphate buffer containing 0.05% of Tween 20

Add 0.5 mL of Tween 20 to 1 I of filtered isotonic phosphate buffer. Mix till complete dissolution. The obtained solution is stable for one week when stored at 4°C.

Preparation of isotonic phosphate buffer containing 0.1% of Tween 20 and 3% BSA

Weigh 3 g of bovine serum albumin (BSA) using laboratory balance and 10 mg of sodium azide using analytical balance, transfer the components to a 125 mL bottle and pour with pre-filtered isotonic phosphate buffer, containing 0.1% of Tween 20 up to 70 mL. Mix to complete dissolution and bring solution to the volume 100 mL. The obtained solution is stable for one month when stored at 4°C.

Note

For standardization of microchips development conditions usage of robotic hybridization station is recommended.

If you have any questions - please contact us: support@semiotik-array.com