DNS Reagent (NY/T Method) - BioReagent, Ready-to-use

Cat. No.: D1528801

CALIDAD & PUREZA BioReagent, Ready-to-use

Synonyms

3,5-Dinitrosalicylic acid reagent

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Application

Cell Metabolism

Descripción general

The carbon nutritional status and quality traits of plants are often assessed using sugar content as an important indicator. Monosaccharides and certain oligosaccharides (e.g., maltose) contain free aldehyde or ketone groups and possess reducing properties, thus they are classified as reducing sugars. Polysaccharides and sucrose, among others, are non-reducing sugars. Taking advantage of the fact that polysaccharides can be hydrolyzed by acid into monosaccharides, the total sugar content can be determined by measuring the monosaccharide content after hydrolysis.

The detection principle of the DNS Reagent (NY/T Method) is that reducing sugars are oxidized to sugar acids under alkaline conditions, while 3,5-dinitrosalicylic acid is reduced to a brownish-red amino compound. Within a certain range, the amount of reducing sugars is proportional to the color intensity of the brownish-red product. The absorbance of the brownish-red substance is measured at 540 nm, and this absorbance value exhibits a linear relationship with the reducing sugar content. The content of reducing sugars and total sugars in samples can be determined using colorimetry and a standard curve.

This reagent is intended for research use only and is not suitable for clinical diagnosis or other applications.

Materials Required (User-Supplied):

1.Distilled water, 6 M hydrochloric acid solution, 6 M sodium hydroxide solution

2.50 mL centrifuge tubes

3.Centrifuge

4.Water bath or constant temperature incubator

5.Spectrophotometer and cuvettes

6.Homogenizer or mortar and pestle

7.I₂-KI solution: Dissolve 5 g of iodine and 10 g of potassium iodide in 100 mL of distilled water.

8.Glucose (Glu) standard solution: 1 mg/mL

Operating Procedure (For Reference Only):

1. Extraction of Reducing Sugars:

① Weigh 0.5–3 g of plant sample, cut into small pieces, add approximately 3 mL of distilled water, and homogenize. Transfer the homogenate to a beaker or Erlenmeyer flask. Rinse the homogenizer 2–3 times with 12 mL of distilled water and combine the washings into the same container.

② Incubate in a 50°C water bath for 30 min with intermittent stirring to ensure complete extraction of reducing sugars.

③ Transfer the precipitate and extract to a 50 mL centrifuge tube and centrifuge at 4000 × g for 5 min.

④ Retain the supernatant. Add 20 mL of distilled water to the precipitate, mix well, and centrifuge again at 4000 × g for 5 min.

⑤ Retain the supernatant. Combine the two supernatants, bring the volume to 100 mL with distilled water (extraction solution), and mix well. This serves as the reducing sugar test solution.

2. Hydrolysis and Extraction of Total Sugars:

① Weigh 0.5–3 g of plant sample, cut into small pieces, add approximately 3 mL of distilled water, and homogenize. Transfer the homogenate to a beaker or Erlenmeyer flask. Rinse the homogenizer 2–3 times with 12 mL of distilled water and combine the washings into the same container.

② Add 10 mL of 6 M hydrochloric acid solution to the container, mix well, and boil for 30 min with intermittent stirring.

③ Spot 2 drops of the hydrolysate onto a glass slide, add 1 drop of indicator solution (I₂-KI solution is recommended; see concentration details under "Materials Required" on the first page of the manual), and check whether hydrolysis is complete. Complete hydrolysis is indicated by the absence of a blue color.

④ After hydrolysis is complete, cool to room temperature. Add 6 M sodium hydroxide solution to adjust the pH to 7.4. Bring the volume to 100 mL with distilled water, mix well, and centrifuge at 4000 × g for 5 min or filter.

⑤ Take 10 mL of the supernatant or filtrate and bring the volume to 100 mL with distilled water to obtain a 10-fold diluted total sugar hydrolysate (extraction solution). Take 1 mL of this total sugar hydrolysate for determination of reducing sugar content.

3. Preparation of Glucose Standard Curve:

Take clean centrifuge tubes or test tubes and proceed according to the table below. Use tube No. 0 as the blank to zero the instrument. Measure the absorbance at 540 nm. Plot the absorbance values on the y-axis against the standard concentrations (mg/mL) on the x-axis to generate the standard curve.

Reagent Added	0	1	2	3	4	5
Glu standard (1 mg/mL)	0	0.2	0.4	0.6	0.8	1
Distilled water (mL)	1	0.8	0.6	0.4	0.2	0
DNS Reagent (mL)	2	2	2	2	2.5	2
Distilled water (mL)	9	9	9	9	9.5	9
Equivalent glucose amount (mg)	0	0.2	0.4	0.6	0.8	1

Note: After adding the DNS reagent, heat in a boiling water bath for exactly 5 minutes, then remove and cool to room temperature under running tap water.

4. Determination of Total Sugars and Reducing Sugars:

Take 1 mL of the prepared reducing sugar extract or total sugar hydrolysate, add 2 mL of DNS Reagent, and proceed with the same operations as for the standard curve. Measure the absorbance of each tube at 540 nm.

Calculation:

Reducing sugar content (%):

Reducing sugar content (%) = (C × V_T) / (m × V_S ) × 100

Total sugar content (%):

Total sugar content (%) = (C × V_T) / (m × V_S) × 100 × 10 × 0.9

C = Sugar amount determined from the standard curve (mg/mL)

V_T = Volume of the extraction solution (mL)

m = Mass of the plant sample (mg)

V_S= Sample volume used for the assay (mL)

Precautions:

1.Avoid repeated freeze-thaw cycles of the above low-temperature reagents and store them protected from light to prevent loss of activity or efficiency.

2.If samples cannot be assayed immediately, store at 2–8°C; they are stable for 3 days.

3.If the reducing sugar concentration of the sample is too high, dilute with distilled water and re-measure, then multiply the result by the dilution factor.

4.The total sugar calculation formula is applicable when interfering impurities are minimal and the reducing sugar content is relatively low compared to the total sugar content. The factor × 0.9 is used to account for the water consumed during hydrolysis when converting total sugars into monosaccharides.

5.Commercially available hydrochloric acid is typically around 12 M and should be diluted to 6 M for use (the exact dilution factor depends on the concentration of the customer's hydrochloric acid. Note that 6 M hydrochloric acid is volatile and should be prepared fresh immediately before use).

6.The reagent should be used as soon as possible after opening to avoid affecting subsequent experimental results.

Especificaciones

Sinónimos

3,5-Dinitrosalicylic acid reagent

Especificaciones y pureza

BioReagent, Ready-to-use

Estabilidad y almacenamiento

Store at 2-8℃ long term (12 months). Store in the dark.

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Protected from light

Enviado en

Wet ice

Este producto requiere envío en cadena de frío. Los servicios terrestres y otros servicios económicos no están disponibles.

Grado

BioReagent, Listo para usar

Product Line

Productos biológicos

Propiedades químicas y físicas

• Sensibilidad: Light-sensitive

Application Protocols

General guidance for a DNS reducing sugar assay aligned with common practice (literature). Validate and adjust to the NY/T method and your matrix; this is not a substitute for the official procedure.

• Materials:

  • DNS Reagent (NY/T Method), distilled/deionized water, reducing sugar standards (e.g., D-glucose), boiling water bath, spectrophotometer set to ~540 nm, glass test tubes or microplate compatible with heating.

• Procedure (cuvette/test tube format):

  1. Prepare a standard curve: e.g., 0–2 mg/mL glucose in water across 6–8 points.
  2. Pipette equal volumes of standard or sample and DNS reagent into tubes (e.g., 1.0 mL + 1.0 mL; follow NY/T if specified differently). Mix.
  3. Heat tubes in a boiling water bath for a fixed time (commonly 5–10 min, method-dependent).
  4. Cool rapidly to room temperature (ice/water). If required, dilute with a fixed volume of water to stabilize color and adjust absorbance into range.
  5. Measure absorbance at ~540 nm against a reagent blank prepared with water instead of sample.
  6. Calculate concentrations from the calibration curve; apply dilution factors. Include QC checks (blank A540, slope/intercept, R2, and a mid-level control).

• Microplate adaptation:

  • Scale volumes proportionally; use heatable plates or pre-heat blocks; ensure uniform heating and edge-effect control.

Note: Exact volumes, heating times, and any stop solution usage are not specified for this item; refer to the NY/T standard and the product insert.

Biological Roles

This product is an analytical reagent; it does not possess intrinsic biological function. However, its analytical target—reducing sugars—play central biological roles.

• Context (general biochemistry):

  • Reducing sugars (e.g., glucose, galactose, maltose) interconvert between cyclic and open-chain forms, the latter bearing an aldehyde/ketone that can reduce DNS under alkaline heat. Their measurement informs on carbohydrate metabolism, enzymatic hydrolysis of polysaccharides, and fermentation performance.
  • In enzymology, DNS-based activity assays quantify rates of glycosidic bond cleavage (e.g., amylases, cellulases, β-galactosidases) via formation of reducing ends.

• Practical implications:

  • DNS assay results are typically reported as “glucose equivalents” based on external calibration; the chromogenic response varies modestly across different sugars (literature), so choice of standard should match the expected predominant analyte when possible.

No clinical or therapeutic claims are associated with this product. It is designated for research use only.

Buffer Applications

Not typically applicable. DNS Reagent (NY/T Method) is a specialized colorimetric reagent, not a buffering system. It is used as supplied for analytical development of color with reducing sugars. For buffer preparation and pH control in related workflows (e.g., enzyme reactions prior to DNS quench), select appropriate biological buffers (e.g., acetate, phosphate, citrate) based on enzyme pH optima and ensure compatibility with subsequent DNS heating steps.

Green Alternatives

DNS assays are widely used but involve alkaline heating and a nitroaromatic dye. Depending on your goals (worker safety, waste minimization, or environmental footprint), consider alternatives.

• Comparison (general, literature-based):
  • DNS (this product):
    • Pros: Robust to many matrices; strong color yield; inexpensive; suitable for enzyme kinetics.
    • Cons: Requires boiling, strongly alkaline; nitroaromatic component; potential interferences with strong reducers; colored waste.
  • 2,4,6-Tripyridyl-s-triazine (TPTZ)/Ferric reducing assays:
    • Pros: Operates at milder temperatures; ferric/ferrous chemistry.
    • Cons: Different specificity (total reducing capacity rather than specific reducing sugars).
  • PAHBAH (p-hydroxybenzoic acid hydrazide) assay:
    • Pros: Often better selectivity for reducing sugars; develops at ~100°C but can offer improved linearity in some matrices.
    • Cons: Hydrazide handling considerations; reagent stability.
  • Phenol–sulfuric acid method:
    • Pros: Broad detection for total carbohydrates (reducing and non-reducing); high sensitivity.
    • Cons: Concentrated H2SO4 hazards; phenol toxicity; strong exotherm.
  • Enzymatic glucose oxidase/peroxidase (GOD/POD):
    • Pros: High specificity for glucose; ambient temperature; amenable to microplate HTS.
    • Cons: Enzyme cost; narrower analyte scope (glucose-centric); requires buffers and peroxidase chromogens.

Selection should weigh hazard profile, analyte specificity, matrix tolerance, and waste stream constraints.

Pharmaceutical Uses

This reagent is not an excipient and is not intended for pharmaceutical formulation. It is supplied for laboratory research and analytical workflows only (Research Use Only).

• Potential roles in pharma/bioprocess labs (non-clinical):
  • In-process control assays to quantify reducing sugars in fermentation broths or enzymatic digests during process development.
  • Screening glycosidase activity in enzyme discovery or QC of carbohydrate-active enzymes.

No pharmacopeial status, dosage form use, or GMP release specifications are provided for this item. For regulated environments, qualify the reagent per your quality system and reference the lot-specific CoA/SDS.

Physical Properties

As supplied, DNS Reagent (NY/T Method) is a formulated mixture; item-specific physical constants are not provided.

• Item-specific properties:

  • Appearance: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular weight, formula (of mixture): Not applicable; mixture.
  • Density, pH, viscosity, refractive index: Not specified for this item; refer to CoA/Spec Sheet.
  • Solubility: Provided as a ready-to-use solution; miscible with water by design (general expectation for DNS assay reagents). Exact specifications not provided.

• Active component (3,5-dinitrosalicylic acid) — literature values for context only (not item specifications):

  • MW 228.12 g/mol; molecular formula C7H4N2O7.
  • Appearance: yellow to orange crystalline solid (literature).
  • Solubility: sparingly soluble in water at neutral pH; freely soluble in alkaline aqueous solutions due to formation of phenolate/carboxylate salts (literature).
  • Typical λmax of reduced chromophore (3-amino-5-nitrosalicylic acid) ~540 nm under standard assay conditions (literature).

Interpretation note: For method development and validation, measure the working reagent’s absorbance blank and stability in your own laboratory setup; rely on the current lot’s CoA for any quantitative specifications.

Quality and Grades

• Supplied grade: BioReagent.

  • Meaning (general): BioReagent grade materials are suitable for common biochemical and molecular biology workflows, emphasizing low levels of contaminants that could interfere with enzymatic or colorimetric assays. They are not necessarily conforming to pharmacopeial monographs and are not intended for clinical/diagnostic use.

• Implications for this reagent:

  • Designed for analytical use in determining reducing sugars via the DNS method (per NY/T standard). Emphasis is typically on consistent chromogenic response, low background absorbance, and batch-to-batch reproducibility.
  • UV/Vis background, metal content, and trace impurities can influence blank absorbance and calibration linearity in colorimetric assays; specific limits are Not specified for this item; refer to CoA/Spec Sheet.

• Stabilizers/inhibitors: Not specified for this item; refer to CoA/Spec Sheet.

• What to expect in practice:

  • Clear labeling of storage (2–8°C) and cold-chain shipping (wet ice) supports stability.
  • Users should verify performance with a standard curve (e.g., glucose, maltose) each run to confirm lot suitability. Record blank A540 and slope/intercept for QC trending.

• Documentation:

  • For regulatory or accredited testing (e.g., per NY/T method), retain the lot CoA and SDS, and document in-house verification data for linear range, LOD/LOQ, and precision.

Reaction and Applications

Primary application: quantification of reducing sugars (e.g., glucose, maltose, lactose, cellobiose) and reducing ends of polysaccharides via the dinitrosalicylic acid (DNS) colorimetric assay aligned with the NY/T method.

• Mechanistic overview (literature):

  • Under strongly alkaline, heated conditions, reducing sugars (in open-chain form) reduce 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid, generating an orange-red chromophore measurable at ~540 nm. Tartrate or other complexants can improve color stability and linearity in some formulations.

• Typical use cases:

  • Enzyme activity assays for glycosidases and amylases (rate of reducing sugar formation).
  • Carbohydrate content in biomass hydrolysates, food/feed samples, and fermentation broths.
  • Monitoring saccharification efficiency in lignocellulosic processing.

• Practical tips (general, method-dependent):

  • Use freshly prepared glucose (or relevant sugar) standards covering the expected range; verify linear range for your matrix (often up to ~1–2 mg/mL glucose equivalent; literature guidance).
  • Heat samples with reagent in a boiling water bath; time control is critical (e.g., 5–10 min, literature). Rapid cooling (ice/water) can stabilize color before reading.
  • Blank the spectrophotometer with reagent plus water matrix to compensate for background.
  • Matrix interferences: strong reducing agents (sulfites, ascorbate), colored particulates, or high protein may bias results; consider appropriate controls or cleanup.

• NY/T method alignment:

  • Follow the specific NY/T standard operating parameters (reagent volumes, heating time/temperature, calibration model). Where they differ from common DNS protocols, the NY/T document takes precedence.

Reaction Conditions

Typical DNS assay development conditions (literature guidance; consult the NY/T method for authoritative parameters):

• Reagent/sample mixing:

  • Combine aliquots of aqueous sample or standard with DNS reagent at a fixed ratio (commonly 1:1 v/v or as specified by the method). Vortex to homogenize.

• Color development:

  • Heat in a boiling water bath (≈95–100°C) for a defined period (often 5–10 minutes). Maintain identical timing across standards and unknowns for reproducibility.
  • After heating, rapidly cool in running water or an ice bath to stabilize the chromophore.

• Measurement:

  • Dilute with water if needed to bring absorbance into the linear range; read at ~540 nm against a reagent blank.
  • Construct an external calibration curve using an appropriate reducing sugar standard (e.g., D-glucose) across the working range relevant to your samples.

• Expected performance (general):

  • Linear dynamic range and sensitivity depend on formulation and matrix; verify in-house. Literature reports linearity typically up to ~1–2 mg/mL glucose equivalents in cuvette-based assays, with microplate formats often using lower absolute amounts.

• Interferences and controls:

  • Strong reductants (ascorbate, sulfite), colored particulates, and high protein may bias A540. Use matrix blanks, spike-recovery, or cleanup (e.g., precipitation/filtration) as appropriate.

Note: Exact temperatures, times, and dilution factors for this SKU are not specified; follow the NY/T protocol and the product’s CoA/insert.

Safety and Handling

• GHS information for this specific item: Not specified for this item; refer to the product SDS for authoritative hazard classification, pictograms, and H/P statements.

• General safety considerations for DNS-type reagents (literature/good practice):

  • Composition typically includes an alkaline medium (e.g., NaOH), the nitroaromatic dye (DNS), and often a stabilizer (e.g., Rochelle salt) in some formulations. As a result, the reagent may be corrosive/irritant and harmful if swallowed or inhaled.
  • Heating is required for color development; handle hot alkaline solutions with care to avoid thermal and caustic burns. Use heat-resistant gloves and eye/face protection.
  • Nitroaromatic dyes can stain skin and surfaces; promptly wipe spills with water and appropriate detergent. Avoid contact with strong acids (can release heat) and strong reducing agents beyond intended assay conditions.

• Recommended PPE and controls:

  • Wear lab coat, safety goggles/face shield, and suitable chemical-resistant gloves (e.g., nitrile). Work in a fume hood when heating or if aerosols/vapors may be generated.
  • Use heat-resistant glassware; do not tightly cap tubes during boiling to prevent pressure buildup.

• First aid (general guidance):

  • Skin/eye contact: Immediately flush with water for ≥15 min; remove contaminated clothing. Seek medical attention for persistent irritation.
  • Ingestion/inhalation: Rinse mouth; move to fresh air; seek medical attention. Do not induce vomiting unless directed by medical personnel.

Always consult the SDS for this SKU before use and for waste disposal guidance in accordance with local regulations.

Solvent Selection

This product is a ready-to-use aqueous reagent solution for colorimetric analysis and is not intended to be used as a reaction solvent.

• Practical notes:

  • The reagent is designed for direct mixing with aqueous sample matrices or hydrolysates. If samples contain significant organic solvent, matrix effects can occur; dilute or evaporate organics prior to assay.
  • When comparing with other colorimetric systems (e.g., phenol–sulfuric acid), consider compatibility with your sample matrix, heat requirements, and hazard profile.

• Miscibility and polarity:

  • DNS reagents are water-based and fully miscible with water (general expectation). Addition of high levels of organic solvents can precipitate components or change color yield; validate if unavoidable.

For solvent guidance related to synthetic reactions, see non-applicable notes in other sections; this reagent is dedicated to analytical assays rather than as a solvent.

Storage and Reconstitution

• Storage conditions (item-specific): Store at 2–8°C. Maintain cold chain during receipt (shipped on wet ice). Do not freeze unless the CoA/Spec Sheet explicitly permits.

• Stability and appearance:

  • Appearance: Not specified for this item; refer to CoA/Spec Sheet.
  • If crystals or precipitate are observed after cold storage, allow the bottle to warm to room temperature and mix gently to redissolve; if insoluble material persists, filter a small aliquot before use. Document any deviations and verify performance with a standard curve.

• Reconstitution/dilution:

  • Supplied as a ready-to-use reagent. Any concentration adjustments or buffer additions are not specified for this item; follow the NY/T method or internal SOPs if dilution is required. Always revalidate calibration after any modification.

• Handling:

  • Minimize light exposure and repeated heating/cooling cycles. Aliquot if frequent use is expected.
  • Keep container tightly closed to limit evaporation and CO2 ingress, which can alter alkalinity.

• Shelf life: Not specified for this item; refer to CoA/Spec Sheet. For best results, track lot opening date and monitor blank absorbance and standard curve slope as part of routine QC.

Structure and Identity

DNS Reagent (NY/T Method) is a prepared colorimetric reagent mixture used for the quantification of reducing sugars by the dinitrosalicylic acid (DNS) assay per the NY/T protocol. As a formulated reagent, it is a mixture rather than a single, well-defined compound.

• Item identity (this lot):

  • Product name: DNS Reagent (NY/T Method)
  • SKU: D1528801
  • Grade: BioReagent
  • CAS, CID, InChIKey, SMILES: Not applicable to the mixture; not specified for this item.

• Active chromogenic component (literature, for context):

  • 3,5-Dinitrosalicylic acid (DNS; common active dye in DNS reagents)
  • Typical identifiers (literature, not item-specific): CAS 609-99-4; molecular formula C7H4N2O7; MW 228.12 g/mol; common functional groups: salicylic acid core bearing two nitro substituents (3,5-dinitro) and a phenolic OH and carboxylic acid.
  • Structural features (literature): Aromatic ring with ortho-hydroxyl and carboxylate capable of intramolecular H-bonding; strongly electron-withdrawing nitro groups that render the ring susceptible to reduction under alkaline, heated conditions.

• 2D structural description (general): The active dye is a substituted salicylic acid: a benzene ring bearing OH at C2, CO2H at C1, and NO2 groups at C3 and C5. Under assay conditions, DNS is reduced to 3-amino-5-nitrosalicylic acid, producing an orange-red chromophore.

Note: Exact formulation constituents and concentrations for this product are not specified; refer to the CoA/Spec Sheet for mixture composition.

Synthetic Utility

Not typically applicable. DNS Reagent (NY/T Method) is formulated for analytical colorimetry rather than for use as a synthetic reagent or building block.

• Related chemistry insight (general):
  • The active dye (3,5-dinitrosalicylic acid) is a nitroaromatic that undergoes reduction under strongly basic, heated conditions—a transformation leveraged analytically, not synthetically, in this context.

For synthetic applications involving nitroaromatic reductions or phenolic acids, use dedicated reagents and protocols; the DNS reagent mixture is not intended for such transformations.

Target Specificity

Not applicable. This product is not a biological targeting reagent (e.g., antibody, enzyme inhibitor). In the analytical sense, the DNS method responds to compounds with reducing capacity—particularly reducing sugars capable of forming an open-chain aldehyde/ketone under alkaline heat (general literature). Specificity is method-dependent and can vary with analyte structure and matrix.