生物素探针合成翻译成英文

Analysis of User Needs:

Based on the search keyword “生物素探针合成” (Biotin Probe Synthesis), the user is likely a researcher or technician in fields like molecular biology, biochemistry, cell biology, or chemical biology. Their underlying needs can be broken down as follows:

1. Definition and Principle: They want to understand what a biotin probe is and the core principle behind its synthesis and application (i.e., the high-affinity biotin-streptavidin interaction).
2. Synthesis Methods: This is the core need. They seek specific chemical synthesis protocols, steps, and reaction schemes for conjugating biotin to various molecules (like proteins, nucleic acids, small molecules).
3. Reagent and Condition Guidance: They need information on the required reagents (e.g., biotinylation reagents like NHS-biotin, sulfo-NHS-biotin), solvents, buffers, and optimal reaction conditions (pH, temperature, time).
4. Purification and Validation: They are looking for methods to purify the synthesized biotin probe from the reaction mixture and techniques to confirm successful synthesis and quantify the biotin incorporation ratio (e.g., HPLC, mass spectrometry, ELISA-based assays).
5. Application-Specific Design: They might need guidance on choosing the right biotinylation strategy based on the target molecule and the intended application (e.g., pull-down assays, imaging, diagnostic tests).
6. Troubleshooting: They may encounter problems during synthesis (e.g., low yield, non-specific binding) and seek solutions.
7. Latest Advances: They might be interested in novel biotin analogues, “click chemistry” approaches, or cleavable biotin probes for advanced applications.

A Comprehensive Guide to Biotin Probe Synthesis: From Principle to Application

The term “Biotin Probe Synthesis” refers to the chemical process of attaching a biotin molecule to a target biological molecule (like a protein, antibody, nucleic acid, or drug compound), creating a powerful tool for detection, isolation, and analysis. This guide is designed to comprehensively address all aspects of this crucial laboratory technique.

1. The Core Principle: Why Biotin?

Biotin (Vitamin B7) has an extraordinarily high affinity for streptavidin or avidin (Kd ≈ 10⁻¹⁵ M), one of the strongest non-covalent interactions in nature. By synthesizing a biotin probe, you effectively “tag” your molecule of interest. This tag can then be recognized by enzyme-conjugated, fluorescently-labeled, or bead-immobilized streptavidin, enabling a wide range of downstream applications.

2. Key Synthesis Methods and Strategies

The synthesis method depends heavily on the target molecule you wish to biotinylate.

A. Protein Biotinylation:
This is the most common application. It typically involves reacting amine-reactive biotin esters with lysine residues on the protein surface.

• NHS-Biotin: The standard reagent for organic solvent-based reactions.
• Sulfo-NHS-Biotin: A water-soluble, membrane-impermeant version that is ideal for labeling surface proteins of live cells without cell penetration.
• Protocol Outline:
  1. Prepare: Dissolve the protein in a suitable buffer (e.g., PBS, pH 7.2-8.5). Avoid amine-containing buffers like Tris.
  2. React: Add a molar excess of the NHS-biotin reagent to the protein solution. The excess ensures efficient labeling.
  3. Incubate: Allow the reaction to proceed for 30 minutes to 2 hours at room temperature or 4°C.
  4. Quench & Purify: Stop the reaction by adding excess lysine or glycine. Purify the biotinylated protein from unreacted biotin using dialysis, gel filtration, or desalting columns.

B. Nucleic Acid Biotinylation:
Biotin can be incorporated into DNA or RNA.

• Enzymatic Labeling: Using DNA polymerases or RNA polymerases with biotin-labeled nucleotides (e.g., biotin-dUTP) during PCR or in vitro transcription.
• Chemical Labeling: Using reactive biotin compounds to modify specific bases (e.g., cytosine) in oligonucleotides.

C. Chemical Conjugation for Small Molecules:
For small molecules or other ligands without primary amines, other biotinylation reagents are available:

• Thiol-Reactive Biotin: Targets cysteine sulfhydryl groups (e.g., maleimide-biotin, biotin-HPDP).
• Carbohydrate Biotinylation: Targets oxidized sugar groups on glycoproteins (using hydrazide-biotin).
• "Click Chemistry": A modern, highly efficient approach using bio-orthogonal reactions like the copper-catalyzed azide-alkyne cycloaddition. An alkyne-biotin can be “clicked” to an azide-modified molecule, or vice-versa.

3. Purification and Validation: Confirming Success

Synthesis is only half the battle; you must confirm you have a functional probe.

• Purification: Remove unincorporated biotin using size-exclusion chromatography, dialysis, or precipitation methods.
• Validation:
  • Gel Shift Assay: Biotinylated proteins may show a slight shift in mobility on a gel when incubated with streptavidin.
  • Western Blot: Run the probe on a gel, transfer to a membrane, and detect with streptavidin-HRP.
  • HPLC/MS: Provides precise confirmation of molecular weight and purity.
  • Functional Assay (e.g., ELISA): Test the probe’s ability to bind its target and then be detected by streptavidin-ELISA reagent.

4. Choosing the Right Strategy: A Practical Guide

Select your synthesis path based on your goal:

• For Cell Surface Labeling: Use Sulfo-NHS-Biotin.
• For General Protein Labeling: Use NHS-Biotin.
• For Site-Specific Labeling (e.g., on a cysteine): Use Maleimide-Biotin.
• For Minimal Steric Hindrance: Use a long-arm spacer biotin (e.g., biotin-LC-NHS).
• For Reversible Capture: Use a cleavable biotin reagent (e.g., containing a disulfide bond).

5. Troubleshooting Common Issues

• Low Labeling Efficiency: Increase the molar ratio of biotin reagent to target. Ensure the pH is correct (≥7.0 for amine reactivity).
• Loss of Target Activity: Over-biotinylation can inactivate a protein. Reduce the biotin:protein ratio or try site-specific labeling.
• High Background: Inadequate purification of the probe. Optimize your purification protocol to remove all free biotin.

6. Recent Advances

The field continues to evolve with new tools:

• Fluorescent Biotin Analogs: Combine biotin’s pull-down capability with a fluorescent tag for direct imaging.
• Membrane-Permeant Biotin Probes: For labeling intracellular proteins.
• Bioorthogonal Cleavable Biotin: Allows for gentle elution of captured targets, improving specificity in pull-down assays.

Conclusion