[3dem] questions peptides-Gold Nanoparticle Conjugates protocols: Colloidal Gold or Gold NanoParticle?

Fang Zhao fang.zhao at helsinki.fi
Tue Jul 20 08:12:41 PDT 2010


Dear Denis:

 

If I understand correctly, your "5 nm gold beads" refers 5 nm colloidal gold
in a more accurate way. 

 

If it is colloidal gold, the way to measure gold concentration is to use
absorption 520 nm, formula:  A520 of 1012 /ml particles = 0.005 x r2 . where
r is diameter of the gold particle measured by EM.

 

If it is colloidal gold, their binding to protein/peptide, polysaccharides
are spontaneous and immediate due to their high affinity to these molecules
, you do not need any linker like thio- or S- bonds or any special protocol.

 

The ratio of protein/gold particle based on calculation is not very useful,
as many factors like pH, ionic strength, actual gold particle size
distribution influence the conjugation ratio. A better way is to measure the
minimum stabilizing protein concentration under your actual experimental
condition. It is simple: just prepare a series dilution of your peptide,
let’s say, from 1-10 micro gram per 500 micro liter, add to 500 micro liter
gold solution diluted in a working condition (not important to stick to any
given number as you are trying to find what is the minimum peptide
concentration for this batch of gold solution), measure A580 over all the
dilution tube, and draw a curve to find a point where the curve rise
suddenly. That tube is the minimum concentration of the protein/peptide you
need. 

The method has been used in Immuno Electron microscopy field since 1980s (
See Chapter 8: Particulate Markers for Immunoelectron Microscopy, by John
Lucocoq,  in book “ Fine Structure Immuno-Cytochemistry” ed by Gareth
Griffiths. Springer-Verlag, 1993. You may be able to search Journal article
under these names if book is not immediate available). It is reliable,
plainly understandable, and suitable different gold particle size/protein
peptide combination.

 

For a long time, the term “colloidal gold” is used for the gold suspension
prepared by reduction of AuCl4 salt. Gold ions from the salt is reduced to
atoms and nucleated to form colloidal particles. Under controlled reduction
and stabilizing condition, the particle size between 3.5 to 150 nm can be
achieved , 1.5 to 3.5 nm colloid is possible but more laborious to produce.
The term NanoGold is used for 0.8 nm or 1.4 nm discrete compound gold (not
colloidal gold) from the company NanoProbes.

I have been bearing this concept in mind until today and suddenly found
there is a widespread use of term “gold nanoparticle or Au-NPs” in fields
outside EM, mainly in drug delivery or similar field. If I looked into their
methods, it is inevitably using AuCl4 reduction method, that is colloidal
gold. I googled a non-scientific consumer gold solution company MesoGold,
they called their products plainly as Nanoparticle colloidal gold
(http://www.purestcolloids.com/mesogold.php).

I am just wondering, now the scientific field behaves like fashion, the old
names and methods are re-packed under new fashionable name such as Nano- to
draw more attention. 

 

Hope the above-mentioned information is useful to you.

 

 

Fang

--------------------------------------------------------

Fang Zhao, MD. PhD.

Advanced Microscopy Unit (AMU)

Haartman Institute 

Haartmaninkatu 3

PO Box 21, 00014 University of Helsinki

Finland

Tel. +358 9 1912 6261

E-mail: fang.zhao at helsinki.fi

Web: http://www.hi.helsinki.fi/amu

 

 

-----Original Message-----
From: 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] On
Behalf Of Chaix, Denis
Sent: 19. heinäkuuta 2010 18:58
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] questions peptides-Gold Nanoparticle Conjugates protocols

 

 

Dear All member of this useful mailing list, It's me again I want to
conjugate peptide (17 kDa) on 5 nm gold beads I have found a protocol
"Assembly and Characterization of Biomolecule-Gold Nanoparticle Conjugates
and their Use in Intracellular Imaging" You can find this protocol on this
website
<http://www.springerprotocols.com/Full/doi/10.1385/1-59259-901-X:085?encCode
=VElCOjU4MDpYLTEwOS05NTI5NS0x&tokenString=qn29VLioHz3jRJ+A5557pg>
http://www.springerprotocols.com/Full/doi/10.1385/1-59259-901-X:085?encCode=
VElCOjU4MDpYLTEwOS05NTI5NS0x&tokenString=qn29VLioHz3jRJ+A5557pg==

but I have several questions about this protocol The interesting part of
this protocol is "Attachment of Peptides Directly to Gold Nanoparticles

 

Now my problem with this protocol

1- It's a protocol for 20 nm diameter beads, i plan to solve this problem to
decrease by four the ratio bead peptides (2500:1 to 625:1)

2- In my lab I have access to 5 nm gold beads but i don't know the
cocnentration and in this protocol they advice to use 1 mL of 20-nm-diameter
citrate-capped gold nanoparticles (1.16 nM) to produce a molar ratio of
thiol to gold of 2500:1.

How can I measure the concentration of my gold beads stock solution (maybe
by absorbance?)

3 At the end do you konow an easy method to storage ( store at 4c or
freezing) and characterisation (quantity of peptide conjugates on the beads
etc......) this kind of peptide-gold beads conjugates?

Have you an idiea about this protocol

Do you know an other protocol

 

thank you

 

 

-----------------------------------------------------------------

Chaix, Denis phd

Vanderbilt University

Email:  <mailto:denis.chaix at Vanderbilt.Edu> denis.chaix at Vanderbilt.Edu

Dept. Cell and Developmental
Biology_______________________________________________

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