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Ferrocene NHS Ester |Syn: Ferrocene Carboxylic N-hydroxysuccinimide Ester | Part No. HPT1002

Ferrocene NHS Ester |Syn: Ferrocene Carboxylic N-hydroxysuccinimide Ester | Part No. HPT1002
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Price: $250.00
Availability: In Stock
Model: HPT1002
Manufacturer: Fivephoton Biochemicals

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Ferrocene NHS Ester
 
(Ferrocene Carboxylic N-hydroxysuccinimide Ester)

Highlights: 
  • Highly reactive NHS ester modification reagent to primary amines generating high yields of ferrocene conjugates
  • Widely employed in commercial oligonucleotide synthesis due to its efficacy
  • Since typically conjugated to primary amines with poly-C extensions, steric hindrance is negligable
  • Efficiently conjugated to primary amines on peptides, proteins and modified oligonucleotides
  • Technical assistance is available for conjugation approaches.  Contact technicalsupport@fivephoton.com.
Ferrocene-NHS Ester  Specifications:
Chemical Name:  Ferrocene carboxylic N-hydroxysuccinimide ester
Formula:  C15H13FeNO4
MW:  327.11
Purity:  > 97% 
Absorbance:  Lamdamax:  438 nm,  Extinction Coefficient:  230 M-1 cm-1
Appearance:  Yellow solid
Solubility:  DMSO, Acetonitrile
Storage:  RT, desiccate.  Ships at ambient temperature
 




Cat. No.
Quantity
Price (USD)
HPT1002-0001
10 mg
$250
HPT1002-0005
50 mg
$350
HPT1002-0010
100 mg
$495
HTP1002-0500
500 mg
$1280
HPT1002-0100
1 g
$1880
HPT1002-0700
5 x 10 mg
$350*








 
*Convenient Reaction Pack:  Five vials with 10 mg Ferrocene NHS Ester per vial:  Cost:  $239.00
Please see "Available Options" menu above to order.

 
* Related Ferrocene Modifying Reagents Offered by Fivephoton Biochemicals
 
 

Ferrocene-NHS Ester is an electrochemical active reporter molecule that reacts with primary amines in peptides, proteins and oligonucleotides.  The NHS reactive group allows for conjugation to both alpha and epsilon amino groups in peptide chains, and to free thiol and amines in modified nucleotides.  Conjugation of ferrocene-NHS ester to the epsilon-amino group in lysine provides a multi-carbon linker between the polypeptide backbone and the ferrocene reporter, which limits steric hindrance on intermolecular interactions.  Below is a schematic diagram revealing the reaction between Ferrocene-NHS ester and the epsilon-amino group in lysine, outlining the method of conjugation to peptide chains.  A similar scheme applies toward conjugating to modified oligonucleotides.  Conjugated ferrocene is detected with voltammetry. 

Fig. 1. General Reaction scheme of ferrocene carboxylic NHS Ester with primary amines on biomolecules, forming an amide linkage in the conjugated product:
ferrocene derivates

Examples of applications of ferrocene conjugates as a molecular reporter for peptides and proteins:
1.    Conjugation of ferrocene to antibodies to detect VEGF in serum using voltammetry.1
2.    Detector of protease activity using ferrocene labeled substrate.2
3.    Binding of enzymes to peptide substrates that were conjugated with ferrocene.


References applying Ferrocene as a molecular reporter on peptides:
  1. Biosens Bioelectron. 2010 Mar 15;25(7):1717-22. Epub 2009 Dec 23.1
  2. Anal Chem. 2008 Sep 15;80(18):7056-62. Epub 2008 Aug 16.2
  3. Biosens Bioelectron. 2009 Aug 15;24(12):3524-30. Epub 2009 May 13.
  4. Langmuir. 2010 Mar 23. [Epub ahead of print.
  5. Analyst.  2009 Dec;134(12):2400-4. Epub 2009 Oct 6
   
Protocol:  Peptide Conjugation 
Suspend the peptide (or protein) into a non-amine buffer at pH 8.3 (for example 10 mM Hepes, 150 mM NaCl, pH 8.3) to the highest concentration that the peptide maintains solubility.  Dissolve Ferrocene NHS Ester in DMSO at 100mg/ml, then immediately dispense into the peptide-buffer solution at a 100-1000X molar excess of Ferrocene Ester relative to peptide.  Let the reaction proceed at RT with gentle shaking for 4 hrs.  (If you are conjugating a protein in a cell lysate, use non-amine protease inhibitors and perform the reaction overnight at 4oC with gentle shaking).  Purify the protein-ferrocene conjugate using gel filtration, or desalt in a spin column, collecting the ferrocene-peptide in the void volume.  The Ferrocene-linked peptide will attain a yellowish coloration.  The peptide-ferrocene conjugate can be followed at 438 nm in correspondence with the peptide bond absorbance peak.

 
Protocol:  Oligonucleotide Conjugation
 
Ferrocene-NHS Ester (9.85 mg, 30.11 micromol) is dissolved in 1.0 milliliter of methyl sulfoxide and 3 micromole amino modified oligonucleotide is dissolved in 800 microliter of 0.2 M sodium carbonate buffer (pH 9.5).  The ester solution (400 microliter) is added to the amino-oligonucleotide solution.  The mixture is left for 16 hour at 4oC, after which it is chromatographed on a Sephadex G-25 column using de-ionized water/carbonate buffer (50/50) as eluent.  The fraction with yellow color is dialyzed against water to remove excess salts and unreacted reagents, and then freeze-dried.  The final product is stored in the refrigerator until use.

 
Oligonucleotide Synthesis With Ferrocene Modification Scheme
 

The following provides examples for measuring ferrocene derivatives
  1. Electrochemical experiments are conducted with a CHI 440 electrochemical workstation (CH instruments, Austin, TX) in a conventional three-electrode cell. The working electrodes consist of polycrystalline gold disks with a diameter of 2-mm that are embedded in Kel-F rods. A platinum electrode and an Ag/AgCl electrode are used as the auxiliary and the reference electrodes, respectively. Prior to each measurement, the Au electrodes are polished with diamond paste and alumina slurry down to 0.05 mm on a polished cloth (Buehler, Lake Bluff, IL), followed by sonication in water and ethanol. The electrodes are then rinsed with a copious amount of de-ionized water and dried under a stream of nitrogen. The supporting electrolyte consists of a 0.1 M HClO4 solution. All solutions are prepared with de-ionized water with a water purification system. All electrochemical experiments were conducted under a nitrogen atmosphere at the ambient temperature.
  2. Osteryoung square wave voltammetry: The electrochemistry of ferrocene-double stranded-DNA attached to the polymer membrane was studied with Osteryoung square wave voltammetry using a BAS 100B voltammetric analyzer. The experiments were carried out in 0.2M PBS (pH 6.9) with 0.1M NaCl at room temperature under a nitrogen stream. The Glassy C working electrode was polished with Metadi diamond paste and washed thoroughly with water under sonication before use. A carbon electrode was used instead of a gold electrode because the gold showed an oxidation peak close to the oxidation potential of the ferrocene label under experimental conditions. The electrode surface was modified exactly as described for the gold surface, but using 2 mL of the polymer sample. For the immobilization and hybridization steps, 20 mL each of amino-oligo and Ferrocene-amino-oligo solutions were used. A non-complementary H2N-oligo probe was immobilized and exposed to Ferrocene-oligo following the same procedure described above for the complementary system. The counter and reference electrodes were a Nichrome wire and Ag/AgCl, respectively.
Product Citations
 

Rudewicz-Kowalczyk, Daria, and Iwona Grabowska. (2022).  "Antibody–Ferrocene Conjugates as a Platform for Electro-Chemical Detection of Low-Density Lipoprotein." Molecules 27.17 (2022): 5492.

L Kashefi-Kheyrabadi, HV Nguyen, A Go, et. Al. (2022).  Rapid, multiplexed, and nucleic acid amplification-free detection of SARS-CoV-2 RNA using an electrochemical biosensor. Biosensors and Bioelectron (195), 1 January 2022, 113649  click here

Jagotamoy Das et al , (2021). Reagentless biomolecular analysis using a molecular pendulum.  Nature Chemistry.  volume 13, pages 428–434  click here 

Ribeiro, W. C., L. M. Gonçalves, S. Liébana, M. I. Pividori, and P. R. Bueno. "Molecular conductance of double-stranded DNA evaluated by electrochemical capacitance spectroscopy." Nanoscale (2016).  Link to article:  click here      Link to methods supplement:  click here


References on Applying Ferrocene as a Molecular Reporter on Oligonucleotides, and Voltammatry Applications:
  1. Anne A, Bouchardon A, Moiroux J. 2003. 3'-Ferrocene-labeled oligonucleotide chains end-tethered to gold electrode surfaces: novel model systems for exploring flexibility of short DNA using cyclic voltammetry. J Am Chem Soc 125: 1112-3.
  2. Anne A, Demaille C. 2006. Dynamics of electron transport by elastic bending of short DNA duplexes. Experimental study and quantitative modeling of the cyclic voltammetric behavior of 3'-ferrocenyl DNA end-grafted on gold. J Am Chem Soc 128: 542-57.
  3. Jenkins DM, Chami B, Kreuzer M, Presting G, Alvarez AM, Liaw BY. 2006. Hybridization probe for femtomolar quantification of selected nucleic acid sequences on a disposable electrode. Anal Chem 78: 2314-8.
  4. Munoz-Serrano L, Guadalupe AR, Vega-Bermudez E. 2005. Morphological studies of oligodeoxyribonucleotides probes covalently immobilized at polystyrene modified surfaces. J Biotechnol 118: 233-45.
  5. Radi AE, Acero Sanchez JL, Baldrich E, O'Sullivan CK. 2006. Reagentless, reusable, ultrasensitive electrochemical molecular beacon aptasensor. J Am Chem Soc 128: 117-24.
  6. Wu ZS, Jiang JH, Shen GL, Yu RQ. 2007. Highly sensitive DNA detection and point mutation identification: an electrochemical approach based on the combined use of ligase and reverse molecular beacon. Hum Mutat 28: 630-7.
Storage and Handling Ferrocene-NHS Ester:  Desiccate, RT.
Safety:  Harmful.  Avoid ingestion, skin and eye contact.
Shipping:  Ships at ambient temperature.  Availalbe for international shipping.
kw.  electrochemical oligonucleotide modification conjugate, ferrocene conjugate, electrochemical modification reagent,

 

 

 

 

 


 

Category Download Link
Specification Sheet click here
Conjugation Protocol
 click here
COA click here
MSDS click here

 

 

 



 

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