Biosensor immunoassay silvernanoparticles based on Plasmone resonance scattering development Ling...

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Visual Sandwich Immunoassay System on theBasis of Plasmon Resonance Scattering Signals ofSilver Nanoparticles

Jian Ling, Yuan Fang Li, and Cheng Zhi Huang*

College of Chemistry and Chemical Engineering, and College of Pharmaceutical Sciences,Education Ministry Key Laboratory on Luminescence and Real-Time Analysis, Southwest University,Chongqing 400715, Peoples Republic of China

In this contribution, we established a sandwich immu-

noassay system with a common spectrofluorometer to

collect the plasmon resonance scattering (PRS) signals

from silver nanoparticles (AgNPs) immunotargeted on

glass slides. By taking the immunoreactions of goat

antihuman IgG (Fc fragment specific) antibody (GAH-

IgG), human immunoglobulin (H-IgG), and rabbit antihu-

man IgG (Fab fragment specific) antibody (RAH-IgG) as

an example, we found that if a primary antibody (GAH-

IgG) was first immobilized on the surface of glass slides

and applied to capture target antigen (H-IgG), AgNPs-

labeled secondary antibody (RAH-IgG) could be employed

to detect the target antigen (H-IgG) by forming a sandwich

immune complex on the surface of the glass slide. It was

found that the PRS signals resulting from the AgNPs

immunotargeted on the glass slides could be applied to

the quantitative detection of H-IgG target antigen in the

range of 10-1000 ng/mL with the limit of determination

of 1.46 ng/mL (3) under optimal conditions, which is

sensitive and comparable with reported chemilumines-

cence immunoassays. With a dark-field microscope

coupled with a spectral system, we measured the PRS

features of single AgNPs immunotargeted on the glass

slides, showing that the PRS of single nanoparticles might

have potential applications in analytical chemistry. Fur-

ther findings showed that the strong PRS signals from the

AgNPs immunotargeted on the glass slides can be clearly

seen and distinguished by naked eyes under the excitation

of a common white light-emitting diode (LED) torch.

Therefore, a visual PRS immunoassay system can be

established easily with common glass slides and an LED

torch.

Light scattering signals, which are interference sources in

spectrofluorometry and could be easily detected with a common

spectrofluorometer,1,2 have been found wide applications in the

investigations of the aggregation of chromophores,3,4 characteriza-

tion of nanomaterials,5,6 and detection of bimolecules7-9 and

drugs.10,11 In combination with other techniques such as mic-

roarray,12-14 total internal reflection,15 wavelength ratio-

metry,16,17 angular ratiometry,18 flow injection analysis,19 and

microscopy imaging,20 modified light scattering techniques have

been established with high sensitivity and good selectivity.21,22

Traditionally, organic small molecules (OSMs), especially

chromophores, are mostly employed as light scattering reagents

to detect biomolecules.7,23 With the development of nanotech-

niques, on the other hand, nanomaterials such as gold and silver

nanoparticles (AuNPs, AgNPs) have been used as light scattering

reagents for the detection of biomolecules for their unique optical

properties in the visible spectral range, which originate from the

excitation of the collective oscillations of conducting electrons

known as surface plasmon resonances.24 Generally, monodis-

persed AuNPs with diameter less than 20 nm exhibit weak light

* To whom correspondence should be addressed. Phone: +86-23-68254659.

Fax: +86-23-68866796. E-mail: [email protected].

(1) Pasternack, R. F.; Collings, P. J. Science 1995, 269, 935939.(2) Pasternack, R. F.; Bustamante, C.; Collings, P. J.; Giannetto, A.; Gibbs, E. J.

J. Am. Chem. Soc. 1993, 115, 53935399.

(3) Scolaro, L. M.; Romeo, A.; Pasternack, R. F. J. Am. Chem. Soc. 2004, 126,

71787179.(4) Mazzaglia, A.; Angelini, N.; Darcy, R.; Donohue, R.; Lombardo, D.; Micali,

N.; Sciortino, M. T.; Villari, V.; Scolaro, L. M. Chem.sEur. J. 2003, 9, 5762

5769.(5) He, Y. Q.; Liu, S. P.; Kong, L.; Liu, Z. F. Spectrochim. Acta, Part A 2005,

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297302.(10) Pang, X. B.; Huang, C. Z. J. Pharm. Biomed. Anal. 2004, 35, 185191.(11) Li, N. B.; Luo, H. Q.; Liu, S. P. Talanta 2005, 66, 495500.(12) Bao, P.; Frutos, A. G.; Greef, C.; Lahiri, J.; Muller, U.; Peterson, T. C.;

Warden, L.; Xie, X. Anal. Chem. 2002, 74, 17921797.(13) Wang, Z.; Lee, J.; Cossins, A. R.; Brust, M. Anal. Chem. 2005, 77, 5770

5774.(14) Saviranta, P.; Okon, R.; Brinker, A.; Warashina, M.; Eppinger, J.; Geier-

stanger, B. H. Clin. Chem. 2004, 50, 19071920.

(15) Feng, P.; Shu, W. Q.; Huang, C. Z.; Li, Y. F. Anal. Chem. 2001, 73, 43074312.

(16) Roll, D.; Malicka, J.; Gryczynski, I.; Gryczynski, Z.; Lakowicz, J. R. Anal.

Chem. 2003, 75, 34403445.(17) Aslan, K.; Lakowicz, J. R.; Geddes, C. D. Anal. Chem. 2005, 77, 2007

2014.(18) Aslan, K.; Holley, P.; Davies, L.; Lakowicz, J. R.; Geddes, C. D. J. Am. Chem.

Soc. 2005, 127, 1211512121.(19) Vidal, E.; Palomeque, M. E.; Lista, A. G.; Fernandez Band, B. S. Anal.

Bioanal. Chem. 2003, 376, 38.(20) Huang, C. Z.; Liu, Y.; Wang, Y. H.; Guo, H. P. Anal. Biochem. 2003, 321,

236243.(21) Huang, C. Z.; Li, Y. F. Anal. Chim. Acta 2003, 500, 105117.(22) Ling, J.; Huang, C. Z.; Li, Y. F.; Long, Y. F.; Liao, Q. G. Appl. Spectrosc.

Rev. 2007, 42, 177201.(23) Huang, C. Z.; Li, K. A.; Tong, S. Y. Anal. Chem. 1997, 69, 514520.

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10.1021/ac802152b CCC: $40.75 2009 American Chemical Society 1707Analytical Chemistry, Vol. 81, No. 4, February 15, 2009Published on Web 01/27/2009


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scattering signals when measured with a common spectrofluo-

rometer, which, however, could be greatly enhanced if the

aggregation of AuNPs occurs. Therefore, light scattering signals

based on the aggregation of AuNPs owing to the interaction of

analytes and biofunctional AuNPs have found applications in the

detection of nucleotide acids,25,26 proteins,27 and drugs28-

30 withhigh sensitivity and simplicity. However, the light scattering signal-

based methods mentioned above suffered from the instability of

the aggregation species in bulk solution, which might bring about

poor stability and reproducibility of light scattering signals from

the aggregates.31

Light scattering signals from metal nanoparticles larger than

40 nm such as AuNPs and AgNPs are colorful without photo-

bleaching in the visual region owing to their unique plasmon

resonance scattering (PRS) properties,32 which are greatly differ-

ent from the scattering signals from the aggregation species of

common OSMs and small nanoparticles. Depending on the size

and shape, the scattered light by AuNPs and AgNPs under the

excitation of a common torch can be orders of magnitude higher

than the emission from fluorescent dyes such as fluorescein.33

The powerful light scattering emission and the adjustable light

scattering color have made promising applications of gold or silver

nanoparticles as light scattering labels in biological imaging and

clinical diagnosis. For example, Schultz et al. employed silver

nanoparticles as immunolabels for in situ hybridization and

immunocytology assays since silver nanoparticles have bright and

nonbleaching PRS properties.34 El-Sayed et al. differentiated

cancer cells from noncancer cells with the light scattering signalsof 40 mm gold nanospheres immunotargeted to epidermal growth

factor receptor (EGFR) overexpressed on cancer cells.35 To our

knowledge, the strong PRS properties of gold and silver nano-

particles have been mainly applied to optical microscopic imaging

of biological cells36,37 and much less frequently applied for

quantitative purpose in terms of analytical chemistry.38

In this contribution, we propose a sandwich-type immunoassay

system with large AuNPs and AgNPs as PRS labels. When the

gold and silver nanolabels are compared under the same condi-

tions, we found that AgNPs, which have much stronger scattering

power than AuNPs, are suitable for the establishment of an

immunoassay system on glass slides. The PRS signals fromAgNPs immunotargeted on the surfaces of glass slides were found

to be easily collected with a common spectrofluorometer, and the

blue PRS light could be easily observed by naked eyes under the

illumination of a white light-emitting diode (LED) torch. In

comparison with the light scattering immunoassay based on the

aggregation of immune-gold or silver we have mentioned above,

our present AgNPs-labeled sandwich immunoassay system, owing

to the direct use of the PRS signals from single AgNPs, avoids

the poor instability and reproducibility of the light scattering

signals from aggregation species.27 Furthermore, our present

system avoids additional steps to reduce silver ions on the surface

of AuNPs to form large silver particles as the light scattering signal

enhancer in comparison to the silver-enhanced gold-labeled

sandwich-type light scattering immunoassays.13,39

EXPERIMENTAL SECTION

Apparatus. The PRS signals were measured with a Hitachi

F-4600 fluorescence spectrophotometer (Tokyo, Japan) by simul-

taneously scanning the excitation and emission monochromator

of the spectrofluorometer with same starting excitation and

emission wavelength (namely, em ) ex). For the detection of

light scattering signals of AgNPs on a glass slide, a very easily

prepared homemade sample holder for the glass slide was

mounted in the sample chamber of the spectrofluorometer (see

the Supporting Information).40The dark-field imaging of AgNPson the glass slides was made with an Olympus BX51 microscope

(Tokyo, Japan), which was equipped with a high numerical dark-(24) Kreibig, U.; Vollmer, M. Optical Properties of Metal Clusters; Springer Verlag:

Heidelberg, Germany, 1995.

(25) Souza, G. R.; Miller, J. H. J. Am. Chem. Soc. 2001, 123, 67346735.(26) Du, B.-A.; Li, Z.-P.; Liu, C.-H. Angew. Chem., Int. Ed. 2006, 45, 81908193.(27) Jiang, Z.; Sun, S.; Liang, A.; Huang, W.; Qin, A. Clin. Chem. 2006, 52,

13891394.(28) Li, Z. P.; Duan, X. R.; Liu, C. H.; Du, B. A. Anal. Biochem. 2006, 351,

1825.(29) Liu, S. P.; Yang, Z.; Liu, Z. F.; Liu, J. T.; Shi, Y. Anal. Chim. Acta 2006,

572, 283289.(30) Liu, Z. D.; Huang, C. Z.; Li, Y. F.; Long, Y. F. Anal. Chim. Acta 2006, 577,

244249.(31) Liu, X.; Dai, Q.; Austin, L.; Coutts, J.; Knowles, G.; Zou, J.; Chen, H.; Huo,

Q. J. Am. Chem. Soc. 2008, 130, 27802782.

(32) Yguerabide, J.; Yguerabide, E. E. Anal. Biochem.1998

, 262, 157176.

(33) Yguerabide, J.; Yguerabide, E. E. Anal. Biochem. 1998, 262, 137156.(34) Schultz, S.; Smith, D. R.; Mock, J. J.; Schultz, D. A. Proc. Natl. Acad. Sci.

U.S.A. 2000, 97, 9961001.(35) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A. Nano Lett. 2005, 5, 829834.(36) Xu, X. H. N.; Chen, J.; Jeffers, R. B.; Kyriacou, S. Nano Lett. 2002, 2, 175

182.(37) Curry, A. C.; Crow, M.; Wax, A. J. Biomed. Opt. 2008, 13, 014022.(38) Taton, T. A.; Lu, G.; Mirkin, C. A. J. Am. Chem. Soc. 2001, 123, 5164

5165.(39) Gupta, S.; Huda, S.; Kilpatrick, P. K.; Velev, O. D. Anal. Chem. 2007, 79,

38103820.

(40) Zhao, H. W.; Huang, C. Z.; Li, Y. F. Anal. Chim. Acta2006

, 564, 166172.

Scheme 1. Preparation Procedures for the PRS

Signal-Based Immunoassay Systema

a The glass slide immobilized with primary antibody (RAH-IgG)was first incubated with antigen (H-IgG) (1) and then incubatedwith AgNPs-labeled secondary antibody (GAH-IgG) (2). After that,the glass slide was transferred for the measurements of the lightscattering signals of AgNPs on a spectrofluorometer or lightscattering imaging on a dark-field microscope (3).

Figure 1. Plasmon resonance scattering spectra of AgNPs immu-notargeted on the glass slide. Concentration of RAH-IgG for im-

mobilization, 2.5 g/mL; incubation time for the capture of antigen

on glass slides, 20 min; AgNPs-antibody (calculated by AgNPs), 1.46

10-11 M.

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field condenser (U-DCW, 1.2-1.4), from which a very narrow

beam of white light can be delivered emitted from a 100 W

tungsten lamp through the bottom of the sample. A 100 /1.3 oil

Iris objective (UPLANFLN, adjustable numerical aperture, from

0.6 to 1.3) was also employed for the dark-field light scattering

imaging. The colorful dark-field light scattering photographs of

AgNPs on the glass slide were captured with a Nikon 4500 digital

camera (Tokyo, Japan). The dark-field light scattering microspec-

tra of single nanoparticles on glass slides were carried out through

the Olympus BX51 dark-field system integrated with an ActonResearch MicroSpec 2300i monochromator and a Princeton

Instruments PI-MAX intensified charge-coupled device (ICCD)

(Trenton, U.S.A.). The size of prepared AgNPs was imaged with

a Hitachi S-4800 scanning electron microscopy (SEM) (Tokyo,

Japan), and the plasmon resonance absorption (PRA) of AgNPs

was measured on a Hitachi U-3010 spectrophotometer (Tokyo,

Japan).

Reagents. Human immunoglobulin (H-IgG), goat antihuman

IgG (Fc fragment specific) antibody (GAH-IgG), rabbit antihuman

IgG (Fab fragment specific) antibody (RAH-IgG), and 3-amino-

propyltriethoxysilane (APTES) were purchased from Sigma-

Aldrich (Missouri, U.S.A.). Bovine serum albumin (BSA) waspurchased from Shanghai Biology Products Institute (Shanghai,

China). Other commercial reagents including glutaraldehyde,

silver nitrate, and trisodium citrate are analytical reagent grade

without further purification. The 0.01 mol/L phosphate buffer

saline (PBS, pH 7.4), prepared by dissolving 0.296 g of

NaH2PO4 2H2O, 2.90 g of Na2HPO4 12H2O, 8.0 g of NaCl, and

1.0 g of KCl in 1 L of ultrapure water (18.2 M), was used as

the buffer for immunoreactions. All water used was ultrapurified

with an LD-50G-E Ultra-Pure water system (Lidi Modern

Waters Equipments Co., Chongqing, China).

Preparation of Gold and Silver Nanoparticles. For the

preparation of large AuNPs,41 50.0 mL of solution containing 0.25

mM of HAuCl4 was prepared in a conical flask, and the pH of

the solution was adjust to 3.5 with 0.1 M HCl. Then, the solution

was brought to boiling while being stirred, and 1.0 mL of 1%

(w/v) trisodium citrate was added to the flask. Under continu-

ous stirring and boiling, the mixture gradually changes to

purple at about 15 min, indicating the formation of large AuNPs.

The colloidal solution of AuNPs was continuous stirred until it

had cooled to room temperature.

Large silver nanoparticles (AgNPs) were prepared according

to the modified Lee-Meisel method.42 Shortly, into a boiling 50.0

mL solution containing 1.0 mM AgNO3 in a conical flask, 2.0

mL of 1% (w/w) trisodium citrate was added. Under continuous

stirring and boiling for about 30 min, the aqueous mixturegradually changes to brown-yellow via yellow, indicating the

formation of large AgNPs. The colloidal solution of AgNPs was

continuous stirred until it had cooled down to room temperature.

It was found that the prepared AuNPs and AgNPs suspension

could be stable at least 3 weeks if stored in a 4 C refrigerator.

The size of AuNPs and AgNPs as prepared were measured with

SEM, and the concentration was calculated according to the

absorbance of the colloidal solution (60nmAgNPs ) 6.75 1010

cm-1 M-1 or 60nmAuNPs ) 5.32 1010 cm-1 M-1).33

Preparation of Antibody-Nanoparticles Conjugates. The

preparation of antibody-AuNPs conjugates was according to the

method reported in refs 43 and 44. Briefly, GAH-IgG (20% more

than the minimum amount, which was determined with a floc-

culation test) was added to the AuNPs suspension of pH 9.0, which

had been previously adjusted with 0.1 M K2CO3 and incubated

at 37 C for about 30 min. The mixture was then centrifuged

at 10 000 rpm for 10 min. After that, the sediment was washed

and resuspended in PBS buffer solution containing 1.0 mg/

mL BSA. The preparation procedures of antibody-AgNPsconjugates are the same as that of antibody-AuNPs.

Procedures. Glass slides (25.4 mm 76.2 mm 1 mm, Shitai

Experimental Instrument Co. Ltd., Haimen, Jiangsu, China) were

cut into 12.7 76.2 pieces, employed herein as the solid support

for immunoassay and were pretreated similarly to common

procedures widely accepted at present for the cleaning and

immobilization of antibody (see the Supporting Information for

details).40 The RAH-IgG antibody-immobilized glass slides were

transferred for immunoreactions into the plastic cell containing

H-IgG in PBS solution for 20 min at 37 C. After being washed

with PBS containing 5 g/mL Tween-20 and ultrapure water three

times, respectively, the glass slides were immersed in the plasticcells containing 1.0 mL of previously prepared AgNPs-antibody

(GAH-IgG) solution for 30 min at 37 C. After the completion of

immunoreactions, the glass slides were washed with water and

dried with nitrogen gas and then transferred for PRS measure-

ments in the spectrofluorometer or imaging on the dark-field

microscope.

Sample Detection. For the detection of H-IgG in human

serum, five serum samples, which were sampled from the Ninth

Peoples Hospital of Chongqing (Chongqing, PRC), were at first

diluted by 105 times with PBS. Then, the primary antibody-

immobilized glass slide was immerged in 1.0 mL of diluted

sample solution for capturing the target of H-IgG. After being

bound with the AgNPs-labeled secondary antibody following

the procedures mentioned above, the glass slides were trans-

ferred for light scattering detection in the spectrofluorometer.

RESULTS AND DISCUSSION

PRS Features of the AgNPs-Labeled Immunoassay Sys-

tem. The principle of this immunoassay is shown in Scheme 1.

The primary antibody (RAH-IgG)-immobilized glass slide was first

employed to capture the target antigen (H-IgG) by incubating with

the antigen, and then, the AgNPs-labeled secondary antibody

(GAH-IgG) was employed in order to form a sandwich immune

complex on the glass slide. Thus, the PRS signals from the AgNPs

immunotargeted on the glass slides could be measured with a

common spectrofluorometer, on which a homemade holder has

been mounted for glass slides in the sample chamber (see the

Supporting Information). As Figure 1 shows, the PRS spectrum

has a characteristic peak located at 410 nm, which is identical to

that of the PRS signals of AgNPs in aqueous medium,45 identifying

that the AgNPs-labeled secondary antibody GAH-IgG have been

really immunotargeted on the glass slides. As could be seen in

(41) Ji, X.; Song, X.; Li, J.; Bai, Y.; Yang, W.; Peng, X. J. Am. Chem. Soc. 2007,

129, 1393913948.

(42) Lee, P. C.; Meisel, D. J. Phys. Chem.1982

, 86, 33913395.

(43) Zhang, C.; Zhang, Z.; Yu, B.; Shi, J.; Zhang, X. Anal. Chem. 2002, 74, 96

99.(44) Ni, J.; Lipert, R. J.; Dawson, G. B.; Porter, M. D. Anal. Chem. 1999, 71,

49034908.

(45) Evanoff, D. D.; Chumanov, G. J. Phys. Chem. B2004

, 108, 1395713962.1709Analytical Chemistry, Vol. 81, No. 4, February 15, 2009


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Figure 1, the PRS signals are dependent on the content of theantigen of H-IgG, indicating that different amounts of antigen can

be distinguished from the PRS signals at 410 nm.

Microscopic Imaging. The microstructure of AgNPs immu-

notargeted on the glass slides could be investigated with SEM

and dark-field microscope, respectively. As Figure 2 shows, the

amount of antibody-AgNPs immunotargeted on the glass slides

depends on the concentration of antigens in the immunoreactions.

The magnified images inset in Figure 2D-F showed that AgNPs

immunotargeted on the glass slides are mainly composed by

nanospheres and there are also a few nanorods (AgNRs), which

should be attributed to the multidistribution of our prepared

AgNPs in size and shape.With the dark-field microscope, we could clearly observe the

light scattering properties of the AgNPs immunotargeted on the

glass slides. Figure 3A shows the PRS images of AgNPs immu-

notargeted on the glass slides, indicating that different AgNPs

display different colors. Corresponding to the colors of these

typical single AgNPs, we could measure the light scattering

spectrum of single AgNPs (Figure 3B). It can be seen that the

one scattering blue light is obviously different from the one

scattering green and red light. The blue ones, characterized about

at 450 nm, are ascribed to the PRS of Ag nanospheres, whereas

the green ones, characterized by two scattering peaks at 450 and

550 nm, and the red ones, characterized by two scattering peaks

at 500 and 630 nm, should be ascribed to that of Ag nanorods. 46

It is known that AgNPs have size- and shape-dependent PRA and

scatterings properties in the spectral range from 400 to 700 nm.45

Therefore, it is easy to understand that AgNPs with different

morphologies immunotargeted on the glass slides should have

different scattering properties.

It should be noted that the difference, in terms of the

characteristic peaks of Figure 1 where the peak is located at 410

nm, and that of Figure 3B, where it is at 450 nm, is possibly

ascribed to the different light sources and detectors between the

fluorescence spectrophotometer and the dark-field microspectro-

scopic system since a xenon lamp and photomultiplier tube (PMT)

detector are employed in the former one, whereas a tungsten lampand ICCD camera are used in the latter one. In addition, it can

be seen from both SEM and the dark-field microscopic images

of AgNPs that the presence of fewer nanorods could not exert a

significant effect on the spectrum of nanospheres immunotargeted

on glass slides.

Comparison with AgNPs and AuNPs Labels. AuNPs with

similar particle size of AgNPs were also used as a PRS labels in

this immune system for comparisons. As Figure 4A shows,

AuNPs-antibody immunotargeted glass slides can be clearly seen

through SEM images, but their light scattering signals are in fact

(46) Link, S.; El-Sayed, M. A. J. Phys. Chem. B1999

, 103, 84108426.

Figure 2. Scanning electron microscopic images of AgNPs immunotargeted on the glass slides. Images from panels A to F show the glass

slides react with H-IgG of different concentrations. The inset squares in panels D, E, and F show the 3-fold multiplied zoom images of thecorresponding circled area. Concentration of RAH-IgG for immobilization, 2.5 g/mL; incubation time for the capture of antigen on glass slides,

20 min; AgNPs-antibody (calculated by AgNPs), 1.46 10-11 M; H-IgG (from A to F), 0, 20, 50, 200, 500, 1000 ng/mL.

Figure 3. Dark-field light scattering features of the immunoassay system. (A) Dark-field light scattering microscopic images of AgNPsimmunotargeted on the antigen-bound glass slides. (B) Plasmon resonance scattering spectra of typical single AgNPs with different scattering

colors in panel A.



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very weak so as to hardly be detected with a spectrofluorometer

under the same experimental conditions as that of AgNPs-labeled

immunoassay (Figure 4B).

With the dark-field microscope to observe scattering light of

AuNPs immunotargeted the glass slides, AuNPs can also be

imaged with a common digital camera (Figure 4C), but it is

compulsory to take much longer exposure time (2 s for AuNPs,

and 0.25 s for AgNPs). In addition, AuNPs immunotargeted on

the glass slide mainly scatter green light, accompanying with some

yellow and red light, which may be ascribed to different size-

dependent PRS properties.47 The PRS spectra of single AuNPs

can also be obtained with dark-field microspectroscopic system

(Figure 4D). It can be seen that the AuNPs scattered green light

has a band characterized at 550 nm, whereas the red one is at

630 nm.

Mie scattering theory,48 which is widely accepted for illustrat-

ing the scattering properties of homogeneous sphere particles with

diameters bigger than about1/20 of the incident wavelength, can

(47) Jain, P. K.; Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A. J. Phys. Chem. B

2006, 110, 72387248.

(48) Mie, G. Ann. Phys.1908

, 25, 377.

Figure 4. Results from the AuNPs-labeled immunoassay. (A) SEM of a AuNPs immunotargeted glass slide. (B) Light scattering spectra of a

AuNPs immunotargeted glass slide. (C) Light scattering image of a single AuNP immunotargeted on a glass slide using dark-field microscopy.(D) Plasmon resonance scattering spectra of a single gold particle in panel C.

Figure 5. Plasmon resonance scattering spectra (A1-C1) and corresponding dark-field light scattering images (A2-C2) of AgNPs with different

sizes immunotargeted on the glass slide. AgNPs size (nm): (A) 35.4; (B) 58.0; (C) 66.0. Experimental conditions: concentration of RAH-IgG forimmobilization, 2.5 g/mL; AgNPs-antibody (calculated by AgNPs), 1.57 10-11 M for 35.4 nm AgNPs, 1.53 10-11 M for 58.0 nm AgNPs,

and 1.52 10-11

M for 66.0 nm AgNPs. Exposure time for capturing the scattering images: (A2) 2 s; (B2 and C2) 0.25 s.

1711Analytical Chemistry, Vol. 81, No. 4, February 15, 2009


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show the different light scattering properties of silver and gold

nanoparticles in the visible region. The Mie scattering cross

section (Csca ), which means the scattering power of a homo-

geneous sphere particle, is expressed as48

Csca)2R2

x2n)1

(2n+ 1)(an2+ bn

2 ) (1)

where R is the particles radius, x is the size parameter given as

2n

medR/, and m is the ratio of refractive index of the sphere

n to that of the surrounding medium nmed. The an and bn in

the right parentheses of eq 1 could be expressed as

an)mn(mx)n(x)-n(x)n(mx)

mn(mx)n(x)-mn(x)n(mx)(2)

bn)n(mx)n(x)-mn(x)n(mx)n(mx)n(x)-mn(x)n(mx)

(3)

wherein n and n are the Riccati-Bessel functions, and the

prime represents first differentiation with respect to the

argument in parentheses.

It can be seen that the light scattering property of a homoge-

neous sphere particle depends on the size and refractive index of

the particle, the incident light wavelength, and the surrounding

medium. Researchers have studied the light scattering of metal

particles in different sizes and components according to the Mie

scattering theory.33,47We can realize that the 60 nm AgNPs have

a maximum Csca when the incident light wavelength is near 420nm, whereas that of the 60 nm AuNPs is near 550 nm, which

explains the reason that 60 nm AgNPs scatter blue light,

whereas 60 nm AuNPs scatter green light. By comparing the

magnitude of maximum Csca reported by refs 45 and 47 we found

that the Csca of 60 nm AgNPs (Csca ) 1.8 10-10 cm2)45 is

approximately 5-fold larger than that of 60 nm AuNPs

(Csca ) 3.5 10-11 cm2),47 indicating that the AgNPs have

stronger scattering power than AuNPs have.

Influence of AgNPs Size. As the size of AgNPs effectively

influences the scattering power of AgNPs (see eq 1), we then

prepared several batches of AgNPs with average diameters of 35.4,

58.0, and 66.0 nm, respectively, and then applied them for further

Figure 6. Immobilization of primary antibody (RAH-IgG) on glassslides. Experimental conditions: H-IgG, 1.0 g/mL; AgNPs-antibody

(calculated by AgNPs), 1.46 10-11 M; incubation time for captureof antigen on glass slides, 20 min; (IPRS), 410 nm.

Figure 7. Effect of incubation time on the capture of antigen on glass

slides. Experimental conditions: concentration of RAH-IgG for im-mobilization, 2.5 g/mL; H-IgG: 1 g/mL; AgNPs-antibody (calculated

by AgNPs), 1.46 10-11 M; (IPRS), 410 nm.

Figure 8. Formation of the sandwich immune structure with of

AgNPs-antibody conjugate. Experimental conditions: concentrationof RAH-IgG for immobilization, 2.5 g/mL; H-IgG: 1 g/mL; incubation

time for the capture of antigen on glass slides, 20 min; (IPRS), 410

nm.

Figure 9. Relationship between bound H-IgG and PRS intensity ofimmunotargeted AgNPs. Concentration of RAH-IgG for immobiliza-

tion, 2.5g/mL; AgNPs-antibody (calculated by AgNPs), 1.46 10-11

M; incubation time for capture of antigen on glass slides, 20 min;

(IPRS), 410 nm.

Table 1. Determination Results of IgG in Five Patients

Sera Samples

sample found (mg/mL) RSD (%, n ) 6) reference (mg/mL)a

1 14.83 11.8 14.172 15.37 7.1 15.20

3 8.79 3.8 6.884 15.50 5.0 16.985 7.79 5.9 8.16

aThe reference results of IgG in patients sera were the clinic reportsmade by Chongqing Ninth Peoples Hospital with immunoturbidimetry.

http://pubs.acs.org/action/showImage?doi=10.1021/ac802152b&iName=master.img-009.png&w=171&h=125http://pubs.acs.org/action/showImage?doi=10.1021/ac802152b&iName=master.img-008.png&w=159&h=124http://pubs.acs.org/action/showImage?doi=10.1021/ac802152b&iName=master.img-007.png&w=167&h=121http://pubs.acs.org/action/showImage?doi=10.1021/ac802152b&iName=master.img-006.png&w=170&h=129


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investigations on the effects of the sizes (see the Supporting

Information). As Figure 5 shows, the scattering signals from the

35.4 nm AgNPs can hardly be distinguished from that of the glass

slide background (Figure 5A1), wether the human IgG is present

or not. However, the 58.0 and 66.0 nm AgNPs, which have

stronger scattering power, can be obviously detected in the

presence of human IgG (Figure 5, parts B1 and C1).

It should be noted that the 66.0 nm AgNPs bound glass slide

cannot show stronger scattering intensity than that of 58.0 nm

AgNPs, although the scattering power of single 66.0 nm AgNPs

is stronger than that of 58 nm AgNPs according to eq 1. As we

know, the scattering intensity of the glass slides bound with

AgNPs is not only dependent on the scattering power of a single

particle but the number of all particles. That might be the reason

that the AgNPs with larger particle size are not of advantage to

bind on glass slides. To confirm this binding assumption for these

AgNPs on the glass slides in the presence of human IgG, dark-

field microscopic observations were made. As the scattering

images in the Figure 5A2-C2 show, all AgNPs with the different

diameters have successfully bound on the surface of the glass

slides, but the glass slide bound with 66.0 nm AgNPs (53 particles/

100 m2) has lower particles density than that of 58.0 nm AgNPs

(67 particles/100 m2).

It can also be seen from the scattering light images of AgNPs

in Figure 5A2-C2 that different sizes of AgNPs scatter different

colors of light, which are accordant with the results as displayed

above (Figure 3) that the single particles with different sizes have

different scattering spectra. Moreover, due to the weak scattering

power, longer exposure time is needed in order to capture the

images of 35.4 nm AgNPs than that of bigger ones (2 s for 35.4

nm AgNPs and 0.25 s for 58.0 and 66.0 nm AgNPs).

Optimization of Experimental Conditions. The immobiliza-

tion of the primary antibody (RAH-IgG) on glass slides is crucial

since it is the first step in the present immunoassay system. The

concentration of primary antibody solution in the immobilization

determines the amount of antibody immobilized on the glass slide.

Series concentrations of antibody solution were tested in order

to investigate the immobilization conditions. After being captured

with sufficient H-IgG and recognized by AgNPs-labeled secondary

antibody completely, these glass slides were investigated by

monitoring the PRS signals of AgNPs immunotargeted on the

glass slides. As can be seen in Figure 6, PRS signals get increased

obviously with the increase of the antibody of RAH-IgG until 2.5

g/mL in the immobilization. Further increase of antibody

concentration in the immobilization does not show much better

results.

The incubation time for capturing antigen by the primary

antibody on the glass slide was also optimized. Figure 7 indicates

that 20 min of incubation was enough. Further incubation with

longer time, for example, longer than 1 h, may destroy the activity

of the antigen and might exert an effect on the reorganization ofAgNPs-labeled secondary antibody with antigen.

The concentration of AgNPs-antibody is another factor af-

fecting the recognition of antigen. Figure 8 shows that AgNPs-

antibody no less that 1.46 10-11 nM (calculated by AgNPs)

should be used in this procedure. Further increase of

AgNPs-antibody concentration did not show obviously better

results.

Determination of Antigen. Figure 9 shows that there is a

good relationship between the enhanced PRS intensity (IPRS)

of glass slides and the concentration of human IgG (cH-IgG) in

solution under the optimal experimental conditions. Human IgG

in the range of 10-1000 ng/mL can be quantitatively detected.The response of PRS signals on the antigen concentration can

be expressed as IPRS ) 216.10 log c -172.45 with R2) 0.995

and the limit of determination of 1.46 ng/mL (3 ). This

sensitivity is comparable with newly reported sensitive chemi-

luminescence immunoassays.49 In order to identify our present

method, five real samples from patient sera (obtained from the

Chongqing Ninth Peoples Hospital, Beibei, Chongqing, PRC)

were detected. As Table 1 shows, the results obtained by our

method are consistent with clinic reports of the hospital by

immunoturbidimetry, indicating that our proposed method is

reliable for clinical diagnosis.

We found that the light scattering signals from AgNPs couldbe clearly seen by naked eyes under a common white LED light

excitation owing to strong PRS emission of AgNPs. Figure 10

exhibits the photographs of six pieces of glass slides directly

captured with a common digital camera under proper illumination

of a white LED torch. The area of glass slides where immunore-

actions occurred clearly scatters blue light due to the immuno-

targeted AgNPs. As can be seen from Figure 10A-F, the

scattering light from the secondary antibody-AgNPs immuno-

targeted on the antigen can be easily distinguished by naked eyes

with increasing antigen content. Consequently, a visual immu-

(49) Fan, A.; Lau, C.; Lu, J. Anal. Chem.2005

, 77, 32383242.

Figure 10. Visual detection of human IgG under the excitation of a white LED torch. Photographs from panels A to F show glass slides bound

with different amounts of IgG. Concentration of RAH-IgG for immobilization, 2.5 g/mL; AgNPs-antibody (calculated by AgNPs), 1.46 10-11

M; H-IgG (from panels A to F), 0, 20, 50, 200, 500, 1000 ng/mL. Incubation time for capture of antigen on glass slides, 20 min. Exposure timefor capturing photograph, 0.125 s.

1713Analytical Chemistry, Vol. 81, No. 4, February 15, 2009
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noassay based on the strong PRS of AgNPs could be established

on common glass slides with simplicity.

CONCLUSION

In this contribution, we propose a PRS immunoassay with

common glass slides as a platform by introducing silver nanopar-

ticles as PRS labels. The light scatting signals of silver nanopar-

ticles could be measured with a common spectrofluorometer for

clinical purposes. On the other hand, the dark-field light scattering

microscopic images showed that single silver nanoparticles canbe clearly seen on the basis of its strong scattering light, indicating

that silver nanoparticles as a light scattering probe may become

a novel model in bioassay. The quantitative study using human

IgG as an antigen showed that the present immunoassay could

have comparable sensitivity with the new reported chemilumi-

nescence immunoassays,41which generally have high sensitivity.

Moreover, the PRS from the AgNPs has different colors depending

on the sizes and shapes, which has potential application in

multiplexed assay using nanoparticles with different scattering

colors.34 The PRS features of a single AgNP, on the other hand,

deserve further investigation and perhaps have potential applica-

tions in analytical chemistry. In addition, the visual immunoassay

system could be constructed and easily observed by naked eyes

with a common LED touch, supplying a new way for visual

detection of immunoreactions on the basis of the light scattering

signals.

ACKNOWLEDGMENT

The authors herein express their great appreciation for the

financial support by the National Natural Science Foundation of

China (Nos. 20425517, 90813019) and the Chongqing Science andTechnology Commission for the Chongqing Key Laboratory on

Luminescence and Real-Time Analysis (2006CA8006).

SUPPORTING INFORMATION AVAILABLE

Additional information as noted in text. This material is

available free of charge via the Internet at http://pubs.acs.org.

Received for review October 10, 2008. AcceptedDecember 27, 2008.

AC802152B


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