Plant total sugar and reducing sugar detection kit (DNS, microcalorimetry)

Cat. No.: P1501777

GRADE & PURITY BioReagent

Overview

The carbon nutritional status in plants and the quality characteristics of agricultural products are often evaluated using sugar content as an important indicator. Monosaccharides and some oligosaccharides (such as maltose) contain free aldehyde or ketone groups, possess reducibility, and are classified as reducing sugars. Polysaccharides and sucrose are non-reducing sugars. The total sugar content can be determined by measuring the monosaccharide content after hydrolysis, utilizing the property that non-reducing sugars can be hydrolyzed to monosaccharides by acid.

Detection Principle: Reducing sugars are oxidized to sugar acids under alkaline heating conditions, while 3,5-dinitrosalicylic acid (DNS) is reduced to a brownish-red amino compound. Within a certain range, the amount of reducing sugar is proportional to the color intensity of the brownish-red product. The absorbance of this brownish-red substance is measured at 540 nm using a microplate reader. This absorbance value has a linear relationship with the reducing sugar content. The reducing sugar and total sugar content in the sample are calculated using a standard curve.

This kit is for scientific research use only and is not intended for clinical diagnosis or other purposes.

P1501777	Component	100T	300T	Storage
P1501777A	Glu Standard (1 mg/mL)	1 mL	1 mL	2-8℃
P1501777B	DNS Detection Solution	10 mL	30 mL	RT. Store in the dark.
P1501777C	Color Solution (for Total Sugar)	5 mL	10 mL	RT. Store in the dark.

User-Prepared Instruments and Reagents

1. Distilled water, Hydrochloric acid solution, Sodium hydroxide solution

2. 50 mL centrifuge tubes, 1 mL centrifuge tubes, Centrifuge, Water bath or incubator, Microplate reader, 96-well plate, Water bath

Experimental Procedure

1. Extraction of Reducing Sugars

1.1 Weigh 0.5-3 g of plant sample, cut into pieces, add about 3 mL of distilled water and homogenize. Transfer to a beaker or conical flask. Rinse the grinder 2-3 times with 12 mL of distilled water and transfer the rinsate to the same container.

1.2 Incubate in a 50°C water bath for 30 min, stirring occasionally to ensure thorough extraction of reducing sugars.

1.3 Transfer the precipitate and extract to a 50 mL centrifuge tube. Centrifuge at 4000 g for 5 min.

1.4 Collect the supernatant. Add 20 mL of distilled water to the precipitate, mix well, and centrifuge again at 4000 g for 5 min.

1.5 Collect the supernatant. Combine the supernatants from the two steps. Dilute to 100 mL with distilled water (this is the extract). Mix well. This serves as the test solution for reducing sugars.

2. Hydrolysis and Extraction of Total Sugars

2.1 Weigh 0.5-3 g of plant sample, cut into pieces, add about 3 mL of distilled water and homogenize. Transfer to a beaker or conical flask. Rinse the grinder 2-3 times with 12 mL of distilled water and transfer the rinsate to the same container.

2.2 Add 10 mL of 6 M hydrochloric acid solution to the container, mix well, then heat in a boiling water bath for 30 min for hydrolysis, stirring occasionally.

2.3 Take 2 drops and place on a glass slide, add 1 drop of Color Solution (about 50 µL) to check if hydrolysis is complete. If hydrolysis is complete, no blue color should develop.

2.4 After hydrolysis, cool to room temperature. Add 6 M sodium hydroxide solution to adjust the pH to 7.4. Dilute to 100 mL with distilled water, mix well. Centrifuge at 4000 g for 5 min or filter.

2.5 Take 10 mL of the supernatant or filtrate and dilute to 100 mL with distilled water, creating a 10-fold diluted total sugar hydrolysate (extract). Take 50 µL of this total sugar hydrolysate to measure its reducing sugar content.

3. Glucose Standard Preparation

Take clean centrifuge tubes or test tubes and prepare a series of Glu standards according to the table below.

Standard Working Solution	Glu Standard (1 mg/mL) (mL)	Distilled Water (mL)	Concentration (mg/mL)
1	0.01	0.04	0.2
2	0.02	0.03	0.4
3	0.03	0.02	0.6
4	0.04	0.01	0.8
5	0.05	0	1.0

4. Assay Setup

Take 1 mL centrifuge tubes. Set up Blank, Standard, and Test wells according to the table below. Add solutions sequentially, avoiding bubbles. Mix carefully. If the sugar concentration in the sample is too high, reduce the sample volume or dilute appropriately before assay. It is best to set up 2-3 replicate wells for samples and take the average.

Reagent (µL)	Blank Well	Standard Well	Test Well
Distilled Water	50	/	/
Glu Standard (1-5)	/	50	/
Extract	/	/	50
DNS Detection Solution	100	100	100

Heat accurately in a boiling water bath for 5 min. Remove, cool to room temperature with tap water. Add 250 µL distilled water.

5. Reducing Sugar Measurement

Mix well. Transfer 300 µL sequentially to the corresponding wells of a 96-well plate. Measure the absorbance of Standard and Test wells at 540 nm, using the Blank well to zero the instrument.

6. Result Calculation

6.1 Standard Curve Plotting

Using the Glu standards (1-5), i.e., the standard glucose concentrations (mg/mL) as the x-axis and the corresponding absorbance values as the y-axis, plot the standard curve. Find the corresponding glucose concentration on the standard curve based on the absorbance of the extract.

6.2 Content Calculation

Percentage Content of Reducing Sugars:

Reducing sugar content per 100 g sample (g) = (c × V _T ) / (m × 1000) × 100 = (c × V _T ) / (m × 10)

Percentage Content of Total Sugars:

Total sugar content per 100 g sample (g) = (c × N × V _T ) / (m × 1000) × 100 × 0.9 = (c × N × V _T ) / (m × 10) × 0.9

Parameter Description

c: Sugar amount found from the standard curve (mg/mL)

V _T : Total volume of the extract, 100 mL

m: Mass of the plant sample, g

N: Dilution factor of the total sugar hydrolysate, 10

Precautions

1. Avoid repeated freeze-thaw cycles for the aforementioned low-temperature reagents to prevent inactivation or decreased efficiency.

2. If test samples cannot be assayed immediately, store at 2-8°C; stable for 3 days.

3. If the sample reducing sugar concentration is too high, dilute with distilled water and re-assay, multiplying the result by the dilution factor.

4. The total sugar calculation formula is used when there are few interfering impurities and the reducing sugar content is relatively small compared to the total sugar content. Multiplying by 0.9 accounts for the water consumed during the hydrolysis of total sugars to monosaccharides.

5. 6 M Hydrochloric Acid Preparation: Generally, commercially available concentrated hydrochloric acid is 11.6-12 M. Mix concentrated hydrochloric acid with distilled or deionized water 1:1 (v/v) to prepare 6 M HCl. Caution: Hydrochloric acid dissolution in water releases heat; handle carefully to avoid injury.

6. 6 M Sodium Hydroxide Preparation: Dissolve 24 g of sodium hydroxide in distilled or deionized water, make up to 100 mL. Caution: Sodium hydroxide dissolution in water releases heat; handle carefully to avoid injury.

7. Use reagents promptly after opening to avoid affecting subsequent experimental results.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions. P1501777A: Store at 2-8℃ long term (12 months). P1501777B, P1501777C: Store at room temperature long term (12 months). Store in the dark.