Overview of Carboxylated
Carboxylated 96-well solid plates are advanced polymer-based microplates engineered for covalent coupling of biomolecules through surface carboxyl (-COOH) groups. These plates are essential for high-throughput binding assays, protein immobilization, nucleic acid conjugation, and enzyme-linked reactions where chemical stability and reproducibility are critical.
Each well contains a solid, activated polymeric surface (typically polystyrene or polypropylene) modified with carboxyl functional groups. The uniform -COOH distribution enables EDC/NHS coupling reactions to covalently attach primary amines from peptides, proteins, or other ligands. For fundamental concepts in chemical bonding and organic functional groups, refer to NIH/NCBI organic chemistry modules (NCBI Bookshelf) and NIST Chemistry WebBook (NIST Chemistry WebBook).
Material Composition and Surface Chemistry
Carboxylated microplates are typically derived from polystyrene substrates, a standard in bioassays for its low autofluorescence and optical clarity. Carboxylation occurs through:
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Plasma polymerization or UV-initiated grafting, introducing COOH groups.
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Chemical oxidation via peroxides or plasma treatment.
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Copolymer incorporation with acrylic acid or methacrylic acid monomers.
The resulting surface presents reactive carboxyl groups that form amide bonds with amine-containing biomolecules after activation. Detailed chemical kinetics for carbodiimide crosslinking reactions can be studied in NIH/NCBI enzymology and bioconjugation resources (NIH Biochemistry Texts) and NIST Surface Chemistry Programs (NIST Surface & Interface Science).
Physical Specifications
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Plate format: 96 wells (standard ANSI/SBS dimensions)
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Material: Polystyrene or polypropylene
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Surface functional group: –COOH (carboxylated)
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Well volume: 350–400 μL (flat bottom)
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Surface area: ~0.32 cm²/well
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Color: Transparent, black, or white (depending on detection mode)
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Compatibility: Suitable for spectrophotometric, fluorometric, or chemiluminescent readouts
Measurement system calibration and dimensional tolerances are guided by NIST Metrology Standards (NIST Dimensional Metrology).
Reaction Principle — EDC/NHS Coupling Mechanism
Carboxylated surfaces require activation before covalent immobilization. The EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) system transforms carboxyl groups into NHS esters, which react readily with primary amines of biomolecules, forming stable amide bonds.
Reaction scheme:
R-COOH+EDC+NHS→R-CO-NHS (active ester)\text{R-COOH} + \text{EDC} + \text{NHS} \rightarrow \text{R-CO-NHS (active ester)} R-CO-NHS+R’-NH₂→R-CO-NH-R’+NHS\text{R-CO-NHS} + \text{R’-NH₂} \rightarrow \text{R-CO-NH-R’} + \text{NHS}
For mechanistic insights into carbodiimide-mediated coupling, see PubChem entries for EDC (CID 2733482) and NHS (CID 7803).
Thermodynamic and kinetic data are further available via NIST Thermochemistry Resources (NIST Thermodynamic Data).
Applications in Research
1. Protein Immobilization
Carboxylated plates enable covalent immobilization of antibodies, enzymes, or antigens, providing enhanced stability and uniformity. These immobilized biomolecules are widely used for:
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Enzyme-linked colorimetric assays
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Surface capture of biomolecules
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High-throughput binding studies
Relevant principles are detailed in NCBI Protein Chemistry Texts (NCBI Biochemistry) and NIH Protein Engineering Initiatives (NIH Research Resources).
2. Peptide and DNA Conjugation
Amine-terminated oligonucleotides or peptides can be covalently attached to carboxylated surfaces for hybridization or molecular screening.
Nucleic acid immobilization protocols are supported by NCBI Genomics Resources (NCBI GenBank) and USGS Biochemical Method Standards (USGS Laboratory Methods).
3. High-Throughput Screening (HTS)
Carboxylated 96-well plates are compatible with robotic liquid handlers, plate readers, and automated imaging systems following ANSI/SBS standards. Engineering standards can be referenced from NIST Metrology for Manufacturing (NIST Manufacturing Metrology).
Optimization Parameters
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pH: Ideal coupling pH 6.0–7.4 for maximal activation.
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Buffer system: MES buffer (0.1 M, pH 6.0) or PBS (pH 7.2). For buffer formulations, refer to PubChem – MES CID 3884.
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Temperature: Room temperature coupling (~22–25 °C).
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Blocking agents: Ethanolamine or BSA to reduce non-specific binding.
Detailed kinetic models of amide bond formation and surface adsorption are discussed in NIST Molecular Surface Science (NIST Surface Chemistry) and NIH Laboratory Chemistry Texts (NIH Chemistry Education).
Storage and Stability
Carboxylated 96-well solid plates should be stored:
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At 4 °C, protected from moisture and light.
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In sealed aluminum pouches or under desiccant.
Storage and shelf-life stability references can be consulted from NIST Chemical Data Service (NIST Chemistry Data) and EPA Laboratory Sample Handling Guidelines (EPA Analytical Guidelines).
Experimental Workflow Example
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Surface Activation: Add 100 µL EDC/NHS solution to each well; incubate 15–30 min.
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Ligand Coupling: Add amine-containing protein (e.g., 50 µg/mL antibody) and incubate 1–2 h at room temperature.
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Blocking Step: Quench residual reactive groups with 1 M ethanolamine, pH 8.5.
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Wash and Dry: Rinse with PBS or deionized water, then dry under sterile air.
For laboratory technique calibration, see NIH Laboratory Practices (NIH Research Resources) and NIST Quality Measurement Practices (NIST Measurement Science).
Quality Control and Validation
QC metrics include:
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Uniform coating confirmation via colorimetric reaction with amine dyes.
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Contact angle measurements to assess hydrophilicity.
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Spectroscopic verification (FTIR or XPS) of –COOH functionalization.
Instrumentation standards and spectroscopic data validation can be found through NIST Spectroscopic Data (NIST Spectroscopy Database) and NIH Analytical Chemistry Education (NIH Analytical Chemistry Texts).
Advantages of Carboxylated Plates
| Parameter | Carboxylated 96-Well Plates | Non-Functional Plates |
|---|---|---|
| Binding Type | Covalent (via amide linkage) | Adsorptive |
| Reproducibility | High | Variable |
| Chemical Stability | Excellent | Moderate |
| Suitable Molecules | Proteins, peptides, oligos | Proteins only |
| Washing Robustness | Strong | Limited |
See NIST Biomolecular Measurement Program (NIST Biomolecular Measurements) for reproducibility frameworks.
Safety and Handling Precautions
Handle EDC/NHS and solvents under chemical hoods using proper PPE.
For comprehensive safety references:
Follow disposal practices per EPA waste management standards (EPA Hazardous Waste Regulations).
Primary keyword: Carboxylated 96-well solid plates
Secondary keywords: –COOH functionalized microplates, EDC/NHS coupling, covalent immobilization surface, protein binding plates, high-throughput 96-well microplate, polystyrene carboxyl surface, enzyme-linked surface assay, solid-phase immobilization, surface chemistry assay plate.
Search intent: laboratory researchers, bioconjugation workflows, surface activation chemistry, high-throughput screening, analytical chemistry instrumentation.