Site-Specific Synthesis and In Situ Immobilization of Fluorescent Silver

Nanoclusters on DNA Nanoscaffolds by Use of the Tollens Reaction

Suchetan Pal, Reji Varghese,, Zhengtao Deng,

Zhao Zhao, Ashok Kumar, Hao Yan, Yan Liu

Department of Chemistry and Biochemistry and

The Biodesign Institute, Arizona State University, (USA)

Angew. Chem. Int. Ed. 2011, 50, 4176 –4179

Ammu Mathew7th May 2011

� Fluorescent silver nanoclusters (AgNCs)

� DNA-templated synthesis of AgNCs

� DNA nanostructures - templates for metallization

� DNA origami structures - superior nanoscale scaffolds

�Tollen’s reagent: commonly employed in carbohydrate chemistry to test for the

aldehyde functionality in reducing sugars

Introduction

Ag2O

aldehyde functionality in reducing sugars

�Site specificity and uniform distribution of the metal NCs along the DNA

templates remain a challenge

DNA-Origami-Directed Self-Assembly of Discrete Silver-Nanoparticle

Architectures

Angew. Chem. Int. Ed. 2010, 49, 2700 –2704

Angew. Chem. Int. Ed. 2011, 50, 2041 –2044

Encapsulation of Gold Nanoparticles in a DNA

Origami Cage

� A new DNA-based method for the synthesis of water-soluble fluorescent AgNCs with a narrow

size distribution by use of the well-known Tollens reaction.

� Site specific synthesis and in situ immobilization of AgNCs on a triangular DNA origami

scaffold.

�The addressability of DNA origami enables the site-specific synthesis and in situ incorporation

of fluorescent AgNCs on the predefined DNA scaffolds with nanometer-scale spatial resolution.

In this paper:

• Small number of sugar moieties were covalently incorporated into a DNA sequence at adjacent

positions.

• Synthesis of AgNCs by the specific stoichiometry of the Tollens reaction: that is, one aldehyde

sugar molecule can reduce two Ag+ ions to Ag02

• These Ag clusters could then act as nucleation sites for further Ag deposition under mild

reductive conditions.

• Tethering of the sugar functional groups to DNA offers stabilization of the AgNCs synthesized.

Strategy:

• DNA1, DNA2,and DNA3, each of which contains 15 nucleotides and one, two, or

three consecutive modified deoxyurinidine (dUm)units, respectively.

Synthesis of Ag NCs on sugar (galactose)-modified DNA strands

(DNA1, DNA2,and DNA3)

Procedure:

Tollens reagent added to the sugar modified DNA (DNA1, DNA2, or

DNA3) in TAE–Mg2+ (tris(hydroxymethyl) aminomethane [Tris]) buffer, and the

mixture was incubated overnight in the dark at room temperature.

each dUm unit carries a sugar unit.

A

B

DNA 1

Ex: 337 nm

Em: 412 nm

3.6 ns (12 %), 0.8 ns (37 %)

and 0.1 ns (51%)

DNA 2

Ex: 337 nm

Em: 411 nm

3.7 ns (12 %), 0.9 ns (42 %)

C

3.7 ns (12 %), 0.9 ns (42 %)

and 0.1 ns (47 %).

DNA 3

Ex: 337 nm

Em: 420 nm

3.8 ns (4 %), 0.9 ns (21 %),

0.3 ns (31 %) and 0.1 ns (44 %)

similar-sized emissive AgNCs with DNA strands carrying different no. of sugar units

a) TEM and b) STEM images of NCs synthesized

by using free DNA3. The inset is a high-

resolution TEM image of a nanocluster. Scale

bars: 10 nm (a and b) and 2 nm (inset of a).

The red shift of the DNA absorbance peak from

260 nm to 275 nm is due to complexation with Ag.

UV-Vis absorption spectra

DNA3 (black) and Tollens reagent (red) ; spectra taken in 30 minutes

interval (for 10 hours) after the addition of 200 fold Ag+.

•DNA 1 and DNA2 similar NCs

in TEM

•Excellent photostability

• No characteristic Ag plasmon

absorption band

• NCs were nearly

monodispersive (~ 2 nm).

Fluorescence emission spectra of DNA3 (5 µM) solutions in different buffer conditions and

Ag+/DNA ratio, after incubation for 12 hours.

1) Fluorescence intensity of the Ag-NC is proportional to Ag+ : sugar modified DNA

2) Effect of Buffer (TAE buffer, HEPES buffer and water)

3) Without the sugar modified DNA as the nucleation site, the fluorescent Ag-NC does not form.

� Formation of Ag NCs involves the initial reduction of Ag+ to Ag0n (n=2, 4, or 6) seed created by

the Tollens reaction by the sugar units , followed by further reduction of extra Ag+ ions by Tris in

the TAE buffer solution

� The maximum size of the cluster would depend on the Ag+/DNA molar ratio, irrespective of the

number of sugar units present on the DNA

Mechanism:

Kinetic evidence : by varying the Tris concentration

Fluorescence emission spectra of DNA3 (5 µM) solutions in different TAEMg2+ buffer concentrations at

fixed Ag+/DNA ratio = 200; after (a)12 hrs (b) 48 hrs, and (c) 96 hrs. Samples were excited at 340 nm.

Origami embellished with silver:

DNA strands with specific sequences and covalently attached sugar moieties were used

for the site-specific incorporation of the sugar units on a DNA origami scaffold. This

approach enabled the subsequent site-specific synthesis and in situ immobilization of

fluorescent Ag clusters at predefined positions on the DNA nanoscaffold by treatment

with the Tollens reagent

Schematic representation of the site-specific immobilization of fluorescent AgNCs on a

triangular DNA origami scaffold

Before After

AFM images of triangular

origami with DNA3 as the

probe Scale bar is 100 nm

and z-scale range is 10 nm.

Histograms showing the

height of the bright

feature on the origami

~ 2.1 nm height difference - apparent diameter of the AgNCs synthesized and deposited in situ

a) Free DNA3

Ex: 337 nm

Em: 420 nm

b) DNA origami

Ex: 340 nm

Em: 418 nm

Free DNA3 DNA origami

DNA2• Fluorescent Ag NCs grown in situ on the origami scaffold had

optical characteristics similar to those of the NCs obtained with

free DNA3.

• No formation of Ag NPs even after several days of incubation.

• No deposition of NCs was observed in other regions of the

triangular DNA origami scaffold, which demonstrates the

excellent site specificity of our DNA templated approach.

a) TEM image of the origami structure after treatment with the

Tollens reagent (the sample was negatively stained with uranyl

formate) and b) corresponding high-resolution TEM image of

the NCs immobilized on the modified arm. c) EDX spectrum of the AgNCs on the DNA origami structure.

Control experiment:

The sugar units act as the nucleation sites for Ag NC formation. The

unmodified DNA scaffolds do not play an active role in the reduction

of the Ag+ ions, but only act as a structural scaffold

A B

(A) AFM images of the triangular shaped DNA origami with one arm carrying ssDNA oligos,

which do not contain the suger modification. No site specific Ag nanocluster formation after

Tollens’ reaction was observed. (B) TEM images of the triangular shaped DNA origami with

one arm carrying ssDNA oligos, which do not contain the suger modification. No site specific

Ag nanocluster formation after Tollens’ reaction was observed. Scale bars are 100 nm.

� A new DNA-based method for the synthesis of water-soluble fluorescent AgNCs through the

use of the well-known Tollens reaction.

� DNA of specific sequences with sugar moieties covalently attached have been successfully

used for the site-specific incorporation of the sugar units on a triangular DNA origami scaffold to

enable the subsequent site-specific synthesis and in situ immobilization of AgNCs at predefined

positions on the DNA nanoscaffold.

Summary

positions on the DNA nanoscaffold.

� The resulting high-density array of emissive NCs may have potential for application in many

fields, such as the fabrication of semiconductor nanostructures.

� Our new approach is characterized by excellent site specific control of NC nucleation and

yields uniformly sized high-density arrays of AgNCs.

� Offers a unique platform for the future site-specific deposition of other metals, such as gold,

which may lead to advances in DNA-based nanoelectronics.

Thank you