We report two novel biomedical applications of Au/Ag alloy nanoparticles called nanocages. In the first
case, composite nanoparticles consisting of a nanocage core and a mesoporous silica shell doped with a photodynamic
sensitizer Yb-2,4-hematoporphyrin were fabricated, characterized, and tested in vitro and ex vivo.
In the second part of the study, Ag nanocubes and Au/Ag nanocages were applied to a multiplexed dot
immunoassay. The assay principle is based on the staining of analyte drops on a nitrocellulose membrane
strip by using multicolor nanoparticles conjugated with biospecific probing molecules.
When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/3493

Khlebtsov, B.N.

Khlebtsov, N.G.

Electronic Sumy State University Institutional Repository

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE NANOMATERIALS: APPLICATIONS AND PROPERTIES Vol. 1 No 2, 02NNBM03(2pp) (2012)   2304-1862/2012/1(2)02NNBM03(2) 02NNBM03-1  2012 Sumy State University Theranostic Applications of Au-Ag Nanocages and Nanocomposites  B.N. Khlebtsov1,*, N.G. Khlebtsov1,2  1 Institute of Biochemistry and Physiology of Plants and Microorganisms,  13, pr. Entuziastov., 410049 Saratov, Russia 2 Saratov State University, 88, Astrachanskaya str., 410026 Saratov, Russia  (Received 26 April 2012; published online16 August 2012)  We report two novel biomedical applications of Au/Ag alloy nanoparticles called nanocages. In the first case, composite nanoparticles consisting of a nanocage core and a mesoporous silica shell doped with a pho-todynamic sensitizer Yb-2,4-hematoporphyrin were fabricated, characterized, and tested in vitro and ex vi-vo. In the second part of the study, Ag nanocubes and Au/Ag nanocages were applied to a multiplexed dot immunoassay. The assay principle is based on the staining of analyte drops on a nitrocellulose membrane strip by using multicolor nanoparticles conjugated with biospecific probing molecules.  Keywords: Nanocomposites, Dot-immunoassay, Nanocages.   PACS numbers: 61.46.Df, 81.16.Be   ______________ * bkh@ibppm.sgu.ru Multifunctional nanoparticles that combine thera-peutic, diagnostic, and sensing modalities are a new trend in nanobiotechnology. One attractive option for theranostic applications are composites that combine the unique optical properties of plasmonic nanoparticles and the mesoporous silica functionalized with an appro-priate photosensitizer (PS). In this work, we fabricated nanocomposites based on silica-coated gold-silver nanocages [1] functionalized with the photodynamic sensitizer Yb-2,4-dimethoxyhematoporphyrin (Yb-HP). The hybrid nanoparticles combine several promising theranostic modalities: (i) an easy tunable plasmon res-onance across the 650-950-nm spectral band with possi-ble use in photothermolysis; (ii) a mesoporous silica shell that preserves the plasmon resonance from an aggregation shift and provides a convenient possibility of surface or volume functionalization with various mo-lecular probes; (iii) a combination of singlet oxygen gen-eration with IR-luminescence band of Yb-HP, which can be used for optically controlled photodynamic therapy [2]. The nanocomposites had uniform distributions over the core and outer shell sizes, were stable in aqueous solution, demonstrated an absorption peak near 400 nm and two fluorescence peaks near 580 and 630 nm, corre-sponding to 4.8×104 bound Yb-HP molecules per one composite particle. Under 630 nm excitation, the com-posites generated singlet oxygen and produced heat under laser irradiation at the plasmon resonance wave-length (750-800 nm). The visible fluorescence, IR-luminescence, and singlet oxygen generation of bound Yb-HP molecules were not quenched owing to metal-PS interaction because of the separation from the nanocage core by the mesoporous silica shell. In particular, we observed enhanced killing of HeLa cells incubated with nanocomposites and irradiated by 630 nm light. An ad-ditional advantage of fabricated conjugates was an IR-luminescence band (900-1060 nm), originating from Yb3+ ions of bound Yb-HP and located in the long-wavelength part of the tissue transparency window. This modality was used to control the accumulation and biodistribution of composite particles in mice bearing Ehrlich carcinoma tumors in a comparative study with intravenously injected free Yb-HP molecules. Thus, the-se multifunctional nanocomposites seem an attractive theranostic platform for simultaneous IR-luminescence diagnostic, photothermal, and photodynamic therapy.    Fig. 1 – Schematic illustration summarizing how fluorescent composite nanoparticles can be fabricated starting with Ag nanocubes and ending with silica-coated Au-Ag nanocages functionalized with Yb-HP molecules. The left-bottom photo shows visible fluorescence of a sample under UV excitation. Designations: EG, ethylene glycol; PVP, poly(vinyl pyrrolidone); IPA, isopropyl alcohol; TEOS, tetraethyl orthosilicate; APTES, 3-aminopropyltriethoxysilane; Yb-HP, Yb-2,4-dimethoxyhe-matoporphyrin; EDC, 1-ethyl-3-(3-dimethylaminopropyl) car-bodiimide; DMSO, dimethyl sulfoxide  Alongside plasmonic nanoparticles, quantum dots (QDs) also serve as popular labels for multicolor optical imaging and immunoassay. However, the potential tox-icity of QDs and the strong need for sample UV irradia-tion are major hurdles toward their widespread applica-tion, particularly in vivo or in nonequipped domestic circumstances (e.g., as a simple immunostrip assay for pregnancy diagnosis). On the other hand, metallic nano-particles are better suited for such applications owing to their ease of synthesis, tunable plasmon resonance, and ready bioconjugation. Most importantly, they are less biotoxic [3] and do not require UV irradiation. Here, we report the first application of Ag nanocubes and multi-color Au/Ag alloy nanoparticles and nanocages to a mul-tiplexed solid-phase dot immunoassay [4]. The assay principle is based on the biospecific staining of analyte  B.N. KHLEBTSOV, N.G. KHLEBTSOV PROC. NAP 1, 02NNBM03(2012)   02NNBM03-2 drops on a nitrocellulose membrane strip by using a mix-ture of multicolor conjugates of nanoparticles functional-ized with molecular probes. Although current synthesis protocols provide a great variety of nanoparticle sizes, shapes, and structures, the Au/Ag alloy nanoparticles seem a convenient platform for the multiplexed dot im-munoassay. Indeed, the galvanic replacement reaction     Fig. 2 – Figure 6 Schematic illustration of the multiplexed dot immunoassay (a, b) and its experimental verification (c). In the first step (panel a), anti-chicken rabbit antibodies Ab1 were spotted in squares A2, B3, C1, D4, and E3; anti-rat rab-bit antibodies Ab2 were spotted in squares A1, B2, C4, D3, and E2; anti-mouse rabbit antibodies Ab3 were spotted in squares A3, B1, C2, and D1; and as a negative control, BSA was spotted in squares A4, B4, C3, D2, and E1. The concentra-tion of all analytes was 100 μg/mL. After staining in a mixture of conjugates (C1 + C2 + C3), the expected spot colors are shown in panel b. The experimental panel c confirms the ex-pected assay results  with silver nanocube templates [1] allows subtle and robust tuning of the Ag/Au conversion ratio and the na-noparticle suspension color from yellow to blue. With these nanoparticles in hand, we were able to conduct a multicolor dot immunoassay in a proof-of-concept exper-iment with three types of molecular targets and biospe-cific probes. Without any instrumental detection, the assay sensitivity was about 20 fmol under naked eye examination. Although this sensitivity is less than that of other techniques (e.g., ELISA, BIOCORE, or other biochemical methods), the simplicity and easy multiplex capability of our multicolor dot assay can find successful application in point-of-care and nonequipped circumstances (e.g., for quick and simple diagnosis of dangerous infections or multiple bacteria under field or domestic conditions). Furthermore, the sensitivity of our dot immunoassay can be improved by using silver enhancement and a flatbed scanner [5].  AKNOWLEDGEMENTS  This work was supported by the Russian Foundation for Basic Research, by grants from the President and the Ministry of Education and Science of the Russian Feder-ation (MK-1057.2011.2, contract no. 14.740.11.260), and by a grant from the Government of the Russian Federa-tion designed to support scientific research projects im-plemented under the supervision of leading scientists at Russian institutions of higher education.   REFERENCES  1. S. Skrabalak, J. Chen, Y. Sun, X. Lu, L. Au, C.M. Cobley, Y. Xia, Acc. Chem. Res. 41, 1587 (2008). 2. B. Khlebtsov, E. Panfilova, V. Khanadeev, O. Bibikova, G. Terentyuk, A. Ivanov, V. Rumyantseva, I. Shilov, A. Ryabova, V. Loshchenov, N. Khlebtsov,  ACS Nano, 5, 7077 (2011). 3. E. Panfilova, A. Shirokov, B. Khlebtsov, L. Matora and N. Khlebtsov, Nano Res., 2012 (in press). 4. N.G. Khlebtsov, L.A. Dykman, Chem. Soc. Rev. 41, 1647 (2011). 5.  C.-H Yeh, C.-Y. Hung, T.C. Chang, H.-P. Lin, Y.-C. Lin, Microfluid Nanofluid  6, 85, (2009).  

Theranostic Applications of Au-Ag Nanocages and Nanocomposites

SocioEconomic Challenges Journal (Sumy State University)

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE NANOMATERIALS: APPLICATIONS AND PROPERTIES 
Vol. 1 No 2, 02NNBM03(2pp) (2012) 
 
 
2304-1862/2012/1(2)02NNBM03(2) 02NNBM03-1  2012 Sumy State University 
Theranostic Applications of Au-Ag Nanocages and Nanocomposites 
 
B.N. Khlebtsov1,*, N.G. Khlebtsov1,2 
 
1 Institute of Biochemistry and Physiology of Plants and Microorganisms,  
13, pr. Entuziastov., 410049 Saratov, Russia 
2 Saratov State University, 88, Astrachanskaya str., 410026 Saratov, Russia 
 
(Received 26 April 2012; published online16 August 2012) 
 
We report two novel biomedical applications of Au/Ag alloy nanoparticles called nanocages. In the first 
case, composite nanoparticles consisting of a nanocage core and a mesoporous silica shell doped with a pho-
todynamic sensitizer Yb-2,4-hematoporphyrin were fabricated, characterized, and tested in vitro and ex vi-
vo. In the second part of the study, Ag nanocubes and Au/Ag nanocages were applied to a multiplexed dot 
immunoassay. The assay principle is based on the staining of analyte drops on a nitrocellulose membrane 
strip by using multicolor nanoparticles conjugated with biospecific probing molecules. 
 
Keywords: Nanocomposites, Dot-immunoassay, Nanocages. 
 
 PACS numbers: 61.46.Df, 81.16.Be 
 
 
______________ 
* bkh@ibppm.sgu.ru 
Multifunctional nanoparticles that combine thera-
peutic, diagnostic, and sensing modalities are a new 
trend in nanobiotechnology. One attractive option for 
theranostic applications are composites that combine 
the unique optical properties of plasmonic nanoparticles 
and the mesoporous silica functionalized with an appro-
priate photosensitizer (PS). In this work, we fabricated 
nanocomposites based on silica-coated gold-silver 
nanocages [1] functionalized with the photodynamic 
sensitizer Yb-2,4-dimethoxyhematoporphyrin (Yb-HP). 
The hybrid nanoparticles combine several promising 
theranostic modalities: (i) an easy tunable plasmon res-
onance across the 650-950-nm spectral band with possi-
ble use in photothermolysis; (ii) a mesoporous silica 
shell that preserves the plasmon resonance from an 
aggregation shift and provides a convenient possibility 
of surface or volume functionalization with various mo-
lecular probes; (iii) a combination of singlet oxygen gen-
eration with IR-luminescence band of Yb-HP, which can 
be used for optically controlled photodynamic therapy 
[2]. The nanocomposites had uniform distributions over 
the core and outer shell sizes, were stable in aqueous 
solution, demonstrated an absorption peak near 400 nm 
and two fluorescence peaks near 580 and 630 nm, corre-
sponding to 4.8×104 bound Yb-HP molecules per one 
composite particle. Under 630 nm excitation, the com-
posites generated singlet oxygen and produced heat 
under laser irradiation at the plasmon resonance wave-
length (750-800 nm). The visible fluorescence, IR-
luminescence, and singlet oxygen generation of bound 
Yb-HP molecules were not quenched owing to metal-PS 
interaction because of the separation from the nanocage 
core by the mesoporous silica shell. In particular, we 
observed enhanced killing of HeLa cells incubated with 
nanocomposites and irradiated by 630 nm light. An ad-
ditional advantage of fabricated conjugates was an IR-
luminescence band (900-1060 nm), originating from 
Yb3+ ions of bound Yb-HP and located in the long-
wavelength part of the tissue transparency window. 
This modality was used to control the accumulation and 
biodistribution of composite particles in mice bearing 
Ehrlich carcinoma tumors in a comparative study with 
intravenously injected free Yb-HP molecules. Thus, the-
se multifunctional nanocomposites seem an attractive 
theranostic platform for simultaneous IR-luminescence 
diagnostic, photothermal, and photodynamic therapy. 
 
 
 
Fig. 1 – Schematic illustration summarizing how fluorescent 
composite nanoparticles can be fabricated starting with Ag 
nanocubes and ending with silica-coated Au-Ag nanocages 
functionalized with Yb-HP molecules. The left-bottom photo 
shows visible fluorescence of a sample under UV excitation. 
Designations: EG, ethylene glycol; PVP, poly(vinyl pyrrolidone); 
IPA, isopropyl alcohol; TEOS, tetraethyl orthosilicate; APTES, 
3-aminopropyltriethoxysilane; Yb-HP, Yb-2,4-dimethoxyhe-
matoporphyrin; EDC, 1-ethyl-3-(3-dimethylaminopropyl) car-
bodiimide; DMSO, dimethyl sulfoxide 
 
Alongside plasmonic nanoparticles, quantum dots 
(QDs) also serve as popular labels for multicolor optical 
imaging and immunoassay. However, the potential tox-
icity of QDs and the strong need for sample UV irradia-
tion are major hurdles toward their widespread applica-
tion, particularly in vivo or in nonequipped domestic 
circumstances (e.g., as a simple immunostrip assay for 
pregnancy diagnosis). On the other hand, metallic nano-
particles are better suited for such applications owing to 
their ease of synthesis, tunable plasmon resonance, and 
ready bioconjugation. Most importantly, they are less 
biotoxic [3] and do not require UV irradiation. Here, we 
report the first application of Ag nanocubes and multi-
color Au/Ag alloy nanoparticles and nanocages to a mul-
tiplexed solid-phase dot immunoassay [4]. The assay 
principle is based on the biospecific staining of analyte 
 
B.N. KHLEBTSOV, N.G. KHLEBTSOV PROC. NAP 1, 02NNBM03(2012) 
 
 
02NNBM03-2 
drops on a nitrocellulose membrane strip by using a mix-
ture of multicolor conjugates of nanoparticles functional-
ized with molecular probes. Although current synthesis 
protocols provide a great variety of nanoparticle sizes, 
shapes, and structures, the Au/Ag alloy nanoparticles 
seem a convenient platform for the multiplexed dot im-
munoassay. Indeed, the galvanic replacement reaction  
 
 
 
Fig. 2 – Figure 6 Schematic illustration of the multiplexed dot 
immunoassay (a, b) and its experimental verification (c). In 
the first step (panel a), anti-chicken rabbit antibodies Ab1 
were spotted in squares A2, B3, C1, D4, and E3; anti-rat rab-
bit antibodies Ab2 were spotted in squares A1, B2, C4, D3, 
and E2; anti-mouse rabbit antibodies Ab3 were spotted in 
squares A3, B1, C2, and D1; and as a negative control, BSA 
was spotted in squares A4, B4, C3, D2, and E1. The concentra-
tion of all analytes was 100 μg/mL. After staining in a mixture 
of conjugates (C1 + C2 + C3), the expected spot colors are 
shown in panel b. The experimental panel c confirms the ex-
pected assay results 
 
with silver nanocube templates [1] allows subtle and 
robust tuning of the Ag/Au conversion ratio and the na-
noparticle suspension color from yellow to blue. With 
these nanoparticles in hand, we were able to conduct a 
multicolor dot immunoassay in a proof-of-concept exper-
iment with three types of molecular targets and biospe-
cific probes. Without any instrumental detection, the 
assay sensitivity was about 20 fmol under naked eye 
examination. Although this sensitivity is less than that 
of other techniques (e.g., ELISA, BIOCORE, or other 
biochemical methods), the simplicity and easy 
multiplex capability of our multicolor dot assay can find 
successful application in point-of-care and nonequipped 
circumstances (e.g., for quick and simple diagnosis of 
dangerous infections or multiple bacteria under field or 
domestic conditions). Furthermore, the sensitivity of 
our dot immunoassay can be improved by using silver 
enhancement and a flatbed scanner [5]. 
 
AKNOWLEDGEMENTS 
 
This work was supported by the Russian Foundation 
for Basic Research, by grants from the President and the 
Ministry of Education and Science of the Russian Feder-
ation (MK-1057.2011.2, contract no. 14.740.11.260), and 
by a grant from the Government of the Russian Federa-
tion designed to support scientific research projects im-
plemented under the supervision of leading scientists at 
Russian institutions of higher education.  
 
REFERENCES 
 
1. S. Skrabalak, J. Chen, Y. Sun, X. Lu, L. Au, C.M. Cobley, 
Y. Xia, Acc. Chem. Res. 41, 1587 (2008). 
2. B. Khlebtsov, E. Panfilova, V. Khanadeev, O. Bibikova, 
G. Terentyuk, A. Ivanov, V. Rumyantseva, I. Shilov, 
A. Ryabova, V. Loshchenov, N. Khlebtsov,  ACS Nano, 5, 
7077 (2011). 
3. E. Panfilova, A. Shirokov, B. Khlebtsov, L. Matora and 
N. Khlebtsov, Nano Res., 2012 (in press). 
4. N.G. Khlebtsov, L.A. Dykman, Chem. Soc. Rev. 41, 1647 
(2011). 
5.  C.-H Yeh, C.-Y. Hung, T.C. Chang, H.-P. Lin, Y.-C. Lin, 
Microfluid Nanofluid  6, 85, (2009). 
 


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Theranostic Applications of Au-Ag Nanocages and Nanocomposites

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