TITLE: Measurement of dissolved carbohydrate
AUTHORS: Ying-Yu Hu, Zoe V. Finkel

[DESCRIPTION]
Here we describe a protocol to measure the dissolved carbohydrate, including total dissolved monosaccharides and total dissolved polysaccharides. 
For total dissolved carbohydrate measurement, freeze-dried dissolved carbohydrate samples are initially vortexed in 9 M H2SO4 for 15 s. The solution is diluted for a final H2SO4 molarity of 1.6 M and hydrolyzed for 3 hours at 90 °C. The hydrolysate is alkalinized by adding 12 M NaOH to the hydrolysate, the ratio of [H+] from hydrolysate to [OH-] from NaOH is 0.82. The alkalinized hydrolysate is oxidized by ferricyanide solution. The absorbance of TPTZ-Fe2+ complex is measured in microtiter plate at 595 nm. 
For total dissolved monosaccharide measurement, freeze-dried dissolved carbohydrate samples are alkalinized by 12 M NaOH and then oxidized by ferricyanide solution.  The absorbance of TPTZ-Fe2+ complex is measured in microtiter plate at 595 nm. 
Our method has shown high reproducibility in aldohexoses, ketohexoses, deoxysugars, aldopentoses, uronic acid and amino sugars. The low limit of detection is 0.024 µg C/mL. 


[STEPS]
SECTION: Sample collection
1. GFF filter is combusted for 240 min  at 450 °C 
Glass filter holder is combusted for  120 min  at 500 °C 
Glass filter funnel, flask and 10 mL centrifuge tubes are combusted for  360 min  at 500 °C 
 
Tube caps are acid-washed.
 

SECTION: Sample collection
2. Filter microalgae sample and collect the filtrate, using gentle vacuum pressure (130 mm Hg).
SECTION: Sample collection
3. Transfer 5 mL filtrate into centrifuge tube and flash freeze.
 

SECTION: Sample collection
4. Freeze dry samples before measurement. 
SECTION: Glucose standards
5. Primary standard solution
SECTION: Glucose standards
6. Secondary standard for total dissolved carbohydrate
SECTION: Glucose standards
7. Secondary standard for total dissolved monosaccharide
SECTION: Glucose standards
6.1. Add 45 µL  primary solution into a 2 mL microtube
SECTION: Hydrolysis of total dissolved carbohydrate
8. Prepare water bath 95 °C 
SECTION: Glucose standards
5.1. In a 2 mL microtube, weigh 1 ~ 2 mg D-glucose 
SECTION: Glucose standards
5.2. Add Milli-Q for a final concentration of 1 mg/mL (>600 µL).
SECTION: Glucose standards
6.2. Add 955 µL  Milli-Q  and then vortex for a good mix
SECTION: Glucose standards
6.3.  In 10 mL centrifuge tubes, prepare the following standard solutions:
               uL      MQ  (ul)      H2SO4(ul)      MQ  (ul)      In amber  (ul)      uMC in amber        0      100      100      900      750      0.00        20      80      100      900      750      20.45        40      60      100      900      750      40.91        60      40      100      900      750      61.36        80      20      100      900      750      81.82        100      0      100      900      750      102.27   
 
SECTION: Glucose standards
7.1. Add 10 µL  primary solution into a 2 mL microtube
SECTION: Glucose standards
7.2. Add 990 µL  Milli-Q  and then vortex for a good mix
SECTION: Glucose standards
7.3. In 12 mL amber vials, prepare the following standard solutions:
               uL      MQ  (ul)      H2SO4(ul)      MQ  (ul)      In amber  (ul)      uMC in amber        0      100      100      900      750      0.00        20      80      100      900      750      20.45        40      60      100      900      750      40.91        60      40      100      900      750      61.36        80      20      100      900      750      81.82        100      0      100      900      750      102.27   
 
 

SECTION: Hydrolysis of total dissolved carbohydrate
9. Add 100 µL  Milli-Q  to each tube with freeze-dried sample.

SECTION: Hydrolysis of total dissolved carbohydrate
10. Use reverse pipetting technique, add 100 µL 18 M H2SO4  to standard solution/sample, immediately vortex for 15 s (monitored by timer or stopwatch) 
 

SECTION: Hydrolysis of total dissolved carbohydrate
11. Add 900 µL Milli-Q, tightly cap the centrifuge tube, and vortex for 5 s . 
SECTION: Hydrolysis of total dissolved carbohydrate
12. Place tube into water bath, log the time. 

 

SECTION: Hydrolysis of total dissolved carbohydrate
13. After all samples are in the water bath, reduce temperature to 90 °C .
SECTION: Hydrolysis of total dissolved carbohydrate
14. Label amber vials for TPTZ measurement with white oil based sharpie.

# of vials = # of samples + # of blanks + # of standards
SECTION: Hydrolysis of total dissolved carbohydrate
15. As soon as hydrolysis duration reaches 3 hours, remove the tube from water bath, let it sit in the tap water bath with ice to quickly stop hydrolysis.
SECTION: Hydrolysis of total dissolved carbohydrate
16. Keep all hydrolysate in a dark cabinet at Room temperature . 
SECTION: Prepare TPTZ reagents
17. 12 M NaOH
SECTION: Prepare TPTZ reagents
17.1. Add 15 mL Milli-Q water into a 50 mL Falcon tube.
SECTION: Prepare TPTZ reagents
17.2. Add 12 g NaOH pellet into the water, swirl and have the pellets completely dissolved, let it cool down to Room temperature .
SECTION: Prepare TPTZ reagents
17.3. Transfer the solution into a 25 mL PP volumetric flask, rinse the tube three times by small amount of Milli-Q and combine the rinsed water into flask, top with Milli-Q water to 25 mL.
SECTION: Prepare TPTZ reagents
18. Alkaline solution for potassium ferricyanide
Dissolve 400 mg NaOH and 20 g Na2CO3 in volumetric flask and top to 1 L by Milli-Q. Store at room temperature.
  
 
SECTION: Prepare TPTZ reagents
19. Sodium acetate solution
SECTION: Prepare TPTZ reagents
19.1. Dissolve 164 g sodium acetate, 42 g citric acid and 300 g acetic acid in a 1 L volumetric flask and top to1 L with Mill-Q water.
 
       
SECTION: Prepare TPTZ reagents
19.2. Store at room temperature.
SECTION: Prepare TPTZ reagents
19.3. Dispense solution by serological pipet to avoid having salt precipitated around sealing surface of the bottle.
SECTION: Prepare TPTZ reagents
20. 3 M acetic acid
Weigh 180 g acetic acid in fumehood, transfer the acid into volumetric flask, top to 1 L with Milli-Q water. Store at room temperature.
SECTION: TPTZ method
21. Prepare boiling bath
SECTION: TPTZ method
22. TPTZ reagents
SECTION: TPTZ method
22.1. Potassium ferricyanide (Reagent A)
Weigh 23 mg potassium ferricyanide and transfer into a 100 mL amber reagent bottle. Add 100 mL alkaline solution, vortex until powder is completely dissolved. It is stable for two weeks at room temperature.
  
 

SECTION: TPTZ method
22.2. Ferric chloride (Reagent B)
Ferric chloride hexahydrate is in spherical shape. It is hard to weigh exact 54 mg for a 100 mL solution. Pick a very small ferric chloride ball and log the weight. Transfer the ball into a 100 mL amber reagent bottle. Calculate the acetate solution required. 
Add acetate solution into the amber bottle, vortex until the ball is completely dissolved.

V_acetate = 100 X W_actual/54
 

SECTION: TPTZ method
22.3. TPTZ (Reagent C)

Estimate the total volume required for the assay: 2 mL X (standard # + blank # + sample #)

For each 100 mL TPTZ reagent, weigh and transfer 78 mg TPTZ into an amber reagent bottle, add 100 mL acetic acid solution, vortex until the powder is completely dissolved. 
 
 

SECTION: TPTZ method
23. Total dissolved carbohydrate samples
SECTION: TPTZ method
23.1. Use reverse pipetting technique, transfer 750 µL  hydrolysate of standard/sample to amber vial.
SECTION: TPTZ method
23.2. Add 250 µL  12 M NaOH and vortex.
SECTION: TPTZ method
24. Total dissolved monosaccharide samples
SECTION: TPTZ method
24.1. Add 1200 µL  Milli-Q into the tube with freeze-dried sample

SECTION: TPTZ method
24.2. Use reverse pipetting technique, transfer 984 µL  solution to amber vial.
SECTION: TPTZ method
24.3. Add 16 µL  12 M NaOH and vortex.
SECTION: TPTZ method
25. In a room with dim light, add 1 mL Reagent A into each amber vial.
SECTION: TPTZ method
26. Tightly cap the vial and vortex.
SECTION: TPTZ method
27. Keep in a boiling water bath for 10 min 
SECTION: TPTZ method
28. Remove boiling bath from the heat, keep all vials in the hot water and move them into the room with dim light.
SECTION: TPTZ method
29. Add 1 mL Reagent B and 2 mL Reagent C into the vial and vortex.
SECTION: TPTZ method
30. Shake at Room temperature for 30 min .
SECTION: TPTZ method
31. Under dim light, using reverse pipetting, load 250 uL of blanks, standards, and samples into the microplate (duplicate).
Load column by column. After one column has been loaded,  immediately cover the column with a lid, which has a black membrane on the top to protect sample from light.

 

 

SECTION: UV/VIS spectra (optional)
33. Hydrolysate
SECTION: TPTZ method
32. Read in microplate reader:

Shake for 5 s at 600 rpm in a continuous and high force mode
Read endpoint 595 nm with a measurement time 100 ms
SECTION: UV/VIS spectra (optional)
33.1. Load 200 µL hydrolysate into microplate.

SECTION: UV/VIS spectra (optional)
33.2. Blank: 
Milli-Q : H2SO4 = 10:1
SECTION: UV/VIS spectra (optional)
34. Monosaccharide solutions
SECTION: UV/VIS spectra (optional)
34.1. Load 200 µL solution into microplate.
SECTION: UV/VIS spectra (optional)
34.2. Blank: Milli-Q
SECTION: UV/VIS spectra (optional)
35. Scan UV/VIS spectra from 200 to 400 nm at a step of 1 nm.
SECTION: Calculation
36. Total dissolved carbohydrate
SECTION: Calculation
37. Total dissolved monosaccharide
SECTION: Calculation
38. Total dissolved polysaccharide
  
SECTION: Waste disposal
39. All hydrolysate and TPTZ reagent C need to be neutralized by soda before disposed into the sink.
SECTION: Waste disposal
40. TPTZ reagent B is collected in trace metal waste container.
SECTION: Calculation
36.1. Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each standard for total dissolved carbohydrate.
SECTION: Calculation
36.2. Obtain standard curve by plotting blank subtracted absorbance (Abs') versus carbon (uM C)
  
SECTION: Calculation
36.3. Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each sample
SECTION: Calculation
36.4.   
SECTION: Calculation
37.1. Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each standard for total dissolved monosaccharide.
SECTION: Calculation
37.2. Obtain standard curve by plotting blank subtracted absorbance (Abs') versus carbon (uM C)
   
SECTION: Calculation
37.3. Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each sample
SECTION: Calculation
37.4.