An Indirect Immunoassay for Detecting Antigen Based on Fluorescence Resonance Energy Transfer

doi:10.4236/ajac.2011.24058

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American Journal of Anal yt ical Chemistry, 2011, 2, 484-490

doi:10.4236/ajac.2011.24058 Published Online August 2011 (http://www.SciRP.org/journal/ajac)

An Indirect Immunoassay for Detecting Antigen Based on

Fluorescence Resonance Energy Transfer

Peihui Yang1,2*, Shuguang Yao1, Wei Wei1, Jiye Cai1,2

1Department of Chemistry, Jinan University, Guangzhou, China

2Key Laboratory of Optoelectronic Information and Sensing Technologies of Guangdong Higher Education Institutes,

Jinan University, Guangzhou, China

E-mail: [email protected]

Received March 4, 2011; revised April 29, 2011; accepted May 16, 2011

Abstract

An indirect immunoassay for detecting antigen was developed. It was based on fluorescence resonance en-

ergy transfer (FRET) and quenching of gold nanoparticles. Bovine serum albumin (BSA) was chosen as

model antigen. Fluorescein isothiocyanate (FITC) was attached to anti-BSA antibody (anti-BSA–FITC) as

FRET donor, while BSA was conjugated to gold nanoparticles (GNPs–BSA) as FRET acceptor. The forma-

tion of anti-BSA–BSA immunocomplex resulted in the FRET between anti-BSA–FITC and GNPs–BSA.

Thus, the fluorescence of FRET donor was quenched, and the decreasing fluorescence intensity responded

linearly to the concentration of acceptor within the linear range. The concentration of BSA we obtained ac-

cording to the stoichiometric ratio between BSA and GNPs. Following this approach, we were able to spe-

cifically detect BSA. The detection limit for BSA was 0.5 nM and the linear range of the assay was 2.9 - 43.5 nM.

It had been successfully applied to specific detection of BSA in serum samples.

Keywords: Anti-BSA Antibody, BSA, Gold Nanoparticles, FRET, Indirect Immunoassay

1. Introduction

Over the past few years, nanoprobes have attracted con-

siderable attention in bioassays, and have been exploited

in biodetection, biolabeling and biosensor development

[1-3]. As a new spectral analysis method, fluorescence

resonance energy transfer (FRET) has been widely used

to detection of protein [4-6] and nucleic acid [7], due to

the characteristics of easy operation, high-sensitivity and

fast detection. Surface energy transfer (SET) between

dye molecules and metal nanoparticles has gained inter-

est because this technique is capable of measuring dis-

tances nearly twice as far as FRET [8]. Gold nanoparti-

cles (GNPs) have been of great interest because of their

high extinction coefficient and broad absorption spec-

trum in visible region which is overlapped with the

emission wavelength of usual FRET donor. Both theo-

retical calculations and experimental studies have well

demonstrated that GNPs are a kind of “superquencher”

which can quench the fluorescence of a range of dyes

with extraordinarily high efficiency [9]. Based on this

superquenching effect, GNPs have been successfully

served as acceptor in FRET. Pihlasalo et al. reported a

new and highly sensitive method to detect protein con-

centrations which relying on protein adsorption and

quenching of fluorescently labeled protein. This method

is based on the competitive assay principle with nonco-

valent adsorption of sample protein and dipyrryl-

methene-BF2 530 labeled BSA on nanosized gold parti-

cles in a homogeneous assay format [10]. Mayilo et al.

reported the homogeneous sandwich immunoassay with

GNPs as fluorescence quenchers. A detection limit of

0.7 ng/mL was obtained for protein cardiac troponin T

(cTnT), which is the lowest value reported for a homo-

geneous sandwich assay for cTnT [11]. Biotin assay with

sensitivities in the micromolar range have also been re-

ported [12]. QDs are used in FRET because of several

advantages. Haldar et al. demonstrated a pronounced

effect on the photoluminescence quenching and shorten-

ing of decay time of CdSe QDs during interaction with

GNPs in a GNPs−BSA conjugated CdSe QD system [8].

Yang et al. have attempted to employ two FRET assem-

bles in one system, in which luminescent CdTe QDs as

FRET donors and GNPs serving as the acceptor with

chymotrypsin as the linking bridge [13].

The present work is based on fluorescence signal

P. H. YANG ET AL.485

changes which were induced by antigen. Little attention

has been reported to the indirect immunoassay method

for detecting antigen with gold nanoparticles acting as a

bridge and the quenching of gold nanoparticles to FRET

donor and antigen. Here, we demonstrated a FRET-based

assay system by antigen conjugated gold nanoparticles as

FRET acceptor and FITC attached the corresponding

antibody as FRET donor. By making use of BSA and

anti-BSA antibody as a model antibody–antigen system,

and combining quenching of gold nanoparticles with the

specific recognition between antigen and antibody, we

designed an indirect immunoassay for detecting antigen.

The design rationale for this assay method is illustrated

in Scheme 1.

2. Experimental

2.1. Materials and Methods

BSA with purity above 99% was purchased from

Sigma. The BSA was weighed out and was digested in

PBS buffer. The final concentration of the BSA was 1.3 ×

10–4 M with the molar extinction coefficient of 45900

M–1·cm–1 at 278 nm. Newborn calf serum was purchased

from Hangzhou Sijiqing Biological Engineering Materi-

als Co., Ltd. Anti-BSA antibody was purchased from

Beijing Dingguo Changsheng Biotech. Co., Ltd. FITC,

hemoglobin (purity ≥ 98%) and plasmin were purchased

from Sigma-Aldrich (St. Louis, MO, USA). Glutathione

oxidized was purchased from Sinopharm Chemical Re-

agent Co., Ltd. Chloroauric acid (1.0 × 10–2 M) was pur-

chased from Shanghai Richjoint Chemical Reagents Co.,

Ltd. Sodium citrate solution (1 vol%) was obtained from

Scheme 1. Diagram describing the procedure of FRET: (a)

Labeling anti-BSA antibody with FITC; (b) BSA was con-

jugated to GNPs and quenched by GNPs; (c) FRET oc-

curred upon addition of BSA conjugated GNPs to the an-

ti-BSA–FITC solution. As a result, the fluorescence of the

FITC was quenched by the nearby GNPs, resulting in an

obvious decrease of FITC fluorescence intensity.

Guangzhou Chemical Reagent Factory. Phosphate buffer

(PBS, 0.01 M, pH = 7.4) was prepared according to the

previously reported procedure [14].

All fluorescence spectra were recorded by a Cary Ec-

lipse fluorescence spectrophotometer (Varian, USA), and

absorption spectra were recorded by an UV1901 spec-

trometer (Rui Li Anal. Instr. Com., China). The TEM

images of each sample were obtained by a PHILIPS

TECNAI-10 transmission electron microscope.

ξ-potential measurements were carried out using a Zeta-

sizer3000 H S Malvern Zetasizer (Malvern Instruments

Inc., USA).

2.2. Preparation of Gold Nanoparticles (GNPs)

and Gold Nanorods (GNRs)

GNPs were prepared according to Frens method [15].

Briefly, 5 mL of 1.0 × 10–2 M chloroauric acid was di-

luted to 50 mL with doubled distilled water and brought

to boil. Next, 10 mL of 1% citric acid was added to the

solution. Refluxing of the solution continued until the

color of the boiling solution changes from dark purple to

red vine color. The image clearly shows the nearly

spherical shape of the particles having an average semi-

diameter of 16 ± 2 nm. The concentration of GNPs was

measured according to the absorbance at 520 nm, using

the molar extinction coefficient of 2.7 × 108 M–1·cm–1 [16].

GNRs were prepared using the reported procedure

[17]. TEM image shows as-prepared GNRs with aspect

ratios 4:1.The UV-Vis absorption spectra and TEM im-

age are consistent with the results of Nikoobakgt et al.

[17].

2.3. Labeling Anti-BSA Antibody with FITC

Anti-BSA antibody was labeled by a modification of the

procedure of reported method [18]. Briefly, anti-BSA

antibody (0.2 g/mL) was reacted with FITC (1 mg/mL)

in 0.1 M sodium bicarbonate buffer, for 4 h at 37°C. The

UV-Vis Spectra of various concentration of anti-BSA–

FITC were measured. The stoichiometry for the binding

of FITC to anti-BSA antibody was obtained by the

UV-Vis absorption peak of various concentration of an-

ti-BSA–FITC. The F/P (FITC/protein) ratio was calcu-

lated from the following expression: F/P ratio = 3.1 ×

A495 / [A280 – 0.31 × A495]. The results suggested that an

average of 3.5 molecules of FITC binding to one an-

ti-BSA antibody molecule.

2.4. Preparation of BSA Conjugated Gold Na-

noparticles (GNPs–BSA)

GNPs-BSA was prepared by mixing equal volumes 8.7 ×

10–9 M solution of GNPs with 3.1 × 10–5 M solution of

BSA in PBS buffer. The mixture was incubated at 37°C

P. H. YANG ET AL.

486

overnight.

2.5. Emission Spectrum Measurements

The quenching intensity of gold nanoparticles on BSA

was measured by a Cary Eclipse fluorescence spectro-

photometer at excitation wavelength of 280 nm and

emission wavelength ranging from 300 - 500 nm. Then,

the fluorescence intensity (F) was measured at emission

wavelength of 345 nm; F0 was the intensity of the sys-

tems without GNPs. The fluorescence quenching inten-

sity (ΔF) can be calculated from the following Eqs: ΔF =

F0 – F.

The FRET assays were performed using the fluores-

cence spectrophotometer. By excitation at 480 nm, emis-

sion spectra were in the range of 500 to 700 nm. Then,

the fluorescence intensity (F) of the donor (anti-

-BSA–FITC) was measured at emission wavelength of

522 nm; F0 was the intensity of the systems without the

acceptor (GNPs–BSA). The fluorescence quenching in-

tensity (ΔF) can be calculated from the following Eqs:

ΔF = F0 – F.

2.6. UV-Vis Spectroscopy

UV-Vis spectra were recorded by a UV1901 spectrome-

ter. All UV-Vis spectra were taken in a wavelength range

of 200 - 800 nm.

2.7. Transmission Electron Microscopy

Characterization

Transmission electron micrographs (TEM) were obtained

with a Philips TECNAI-10 microscope. The TEM sam-

ples were prepared by taking a solution sample and cast-

ing it onto a carbon-coated copper grid sample holder

followed by evaporation in air at room temperature.

2.8. Particle Size and Zeta Potential

Measurements

Zeta potential distribution was measured by the particle

characterizer (Malvern ZetaSizer Nano ZS, Malvern,

UK). Briefly, the samples were loaded into the capillary

zeta potential cell for measurement. This instrument also

includes the DLS (dynamic laser scattering) function, by

which the particle size distribution can be analyzed.

2.9. Assay of the BSA in Serum Samples

New-born calf serum was diluted about 100 times during

the disposal procedure. A test of BSA concentration was

analyzed under the optimum concentration of gold na-

noparticles and experimental conditions by standard ad-

dition method.

3. Results and Discussion

3.1. Preparation and Characterization of FRET

Acceptor

In the reported study [19], it was shown that gold

nanoparticles quenched BSA fluorescence mainly

through a static quenching mechanism. The stoichiome-

try for the binding of BSA to GNPs was confirmed by

the relationship between fluorescence quenching inten-

sity and the concentration of GNPs. At pH7.4, upon ad-

dition of GNPs, the fluorescence intensity of BSA at

345 nm decreased with the increase of the concentration

of GNPs. To further increase the concentration of GNPs

produced no obvious change on the fluorescence of BSA,

which indicated that BSA adsorption on GNPs reached

saturation. The stoichiometry for the binding of BSA to

GNPs was calculated by the concentration ratio of BSA

to GNPs, until the fluorescence of BSA had no obvious

change. Varying the concentration of BSA, an average of

3.60 × 103:1 stoichiometry for the binding of BSA to

GNPs was calculated (Table 1). The FRET acceptor was

prepared with the same ratio as BSA to GNPs described

above.

3.2. Spectra Characteristics of FRET System

FRET occurred upon the addition of GNPs–BSA to the

anti-BSA–FITC solution, GNPs–BSA and FITC were

closed due to the specific binding of anti-BSA antibody

and BSA. When GNPs conjugate was present, the fluo-

rescence of FITC was quenched by the FRET acceptor,

as is shown in Figure 1.

Table 1. The stoichiometry for the binding of BSA to GNPs.

nBSA(mol) n GNPs(mol) nBSA:n GNPs nBSA:n GNPs (mean)RSD (%)

1.1 × 10–7

3.0 × 10–11

3.70 × 103

2.0 × 10–7

5.7 × 10–11

3.53 × 103

3.3 × 10–7

9.3 × 10–11

3.53 × 103

3.9 × 10–7

10.7 × 10–11

3.63 × 103

3.60×103

1.7

P. H. YANG ET AL. 487

500 520 540 560 580

100

200

300

400

500

600

700

/nm

Figure 1. The process of FRET between anti-BSA–FTIC

and GNPs–BSA. Emission spectra of anti-BSA–FITC with

the increasing of GNPs–BSA, the concentration of anti-

-BSA–FITC was 0.120 mg/ml and that of GNPs–BSA was

0.9, 1.8, 2.7, 3.6, 4.5, 5.4×10–10 M, respectively.

3.3. Interaction Mechanism between FRET

Donors and FRET Acceptors

3.3.1. Interference of Nonspecific Substances

Interference experiments were performed to exclude

nonspecific interactions of biomolecules, in which GNPs

were incubated with hemoglobin (Hb), oxidized glu-

tathione (GSSG), lysozyme according to the ratio of

BSA to GNPs as the FRET donor, respectively. The re-

sults showed that the capability of recognition with an-

ti-BSA–FITC can be summarized as BSA ＞ Hb ＞

GSSG ＞ lyszyme ＞ GNPs (Figure 2).

The net surface charges of Hb and GSSG at pH 7.4 are

negative, while anti-BSA–FITC is positively charged.

0 15304560

120

160

F

Canalytes×10-9M

BSA

GNPs only

GSSG

Lysozyme

Figure 2. The process of FRET between anti-BSA–FTIC

and GNPs-analyte at 25°C and 0.01 M PBS, and pH value

was 7.4. The concentration of anti-BSA–FITC was 0.120 mg/ml

and that of GNP–analyte was 5.0×10–8 M.

Therefore, the distances between Hb, GSSG and an-

ti-BSA–FITC are shortened due to electrostatic interac-

tions to partially produce FRET, resulting in fluores

cence quenching to a certain extent. The reason why the

quenching effect of Hb is higher than GSSG is that the

Hb has structural similarities to BSA. However, posi-

tively charged lyszyme have little quenching effect.

These results indicated that FRET occurs after recogni-

tion between FRET donor and energy acceptor, resuling

in fluorescence quenching.

3.3.2. Characterizati on of Specific Recog ni ti on

Between FRET Donor and FRET Acceptor

To directly visualize the specific recognition between

FRET donor and FRET acceptor, the gold nanorods with

aspect ratios 4:1 (Figure 3) were conjugated with FRET

donor and incubated with FRET acceptor, and then

dropped cast on a copper grid and examined by TEM

[20]. During imaging, we observed a large amount of

nanoparticle–nanorod dimers as shown in Figure 4,

These nanoparticle dimers are believed to be formed

through specific recognition between antigen and anti-

body. The mean particle size and size distribution were

measured by particle size analyzer [21]. After specific

recognition between antigen and antibody, the average

diameter of gold nanoparticles and nanorods mixed sys-

tem was increased (Figure 5). These results further indi-

cate that FRET occurs after recognition between FRET

donor and FRET acceptor, resulting in fluorescence

quenching.

3.4. Indirect Assay of Antigen with FRET

System

Several parameters such as pH and reaction time need

further optimization for development of this FRET sys

400 500 600 700 800900100011001200

0.0

0.2

0.4

0.6

0.8

1.0

520nm

800nm

Abs

/nm

Figure 3. Absorbance spectra of prepared GNPs with as-

pect ratio 4:1.

P. H. YANG ET AL.

488

Figure 4. TEM micrographs of (a) antigen-conjugated gold nanoparticles; and (b) FRET donor-conjugated gold nanorods:

and (c) nanoparticle-nanorod conjugate oligomers formed from the binding of antigen-conjugated gold nanoparticles and

gold nanorods nanoprobes wi th F RET donor .

(a) (b)

Figure 5. Hydrodynamic diameter distribution plots as determined by DLS measurements: (a) the size distribution of GNPs

and GNRs mixed system; (b) the size distribution of GNPs and GNRs mixed system after specific recognition between antigen

and antibody.

0.0 0.5 1.0 1.5 2.0 2.5

100

150

200

CGNPs-BSA×10-7M

0.12mg/ml

0.075mg/ml

0.038mg/ml

F

Figure 6. The effect of the concentration of donor on the

process of FRET.

tem in order to tailor reaction results. To understand the

effect of the concentration of FRET donor on the process

of FRET, we have studied optimal FRET donor concen-

tration. It was found that the quenching effect increased

as the concentration of FRET donor increasing (Figure

6), which may related to the immunogenicity of FRET

donor. At lower concentrations, the immunogenicity of

FRET donor was low, so, the quenching effect initiated

by energy transfer was weak. As the concentration of

FR-ET donor increased, the immunogenicity of FRET

donor increasing proportionally. Hence, the quenching

effect increased. The quenching effect reached a maxi-

mum when anti-BSA–FITC concentration was 0.12

mg/ml. Therefore, 0.12 mg/ml of GNPs–BSA was used

in the following research.

Since recognition of donor–acceptor requires some time,

the optimal incubating time was studied. For the quenching

of fluorescence intensity of anti-BSA–FITC by GNPs–

BSA, as shown in Figure 7, fluorescence quenching inten-

sity increased gradually at incubating time range from 0

min to 22 min and reached a plateau at 22 min. Thus, 25

min was chosen for the assay. The influence of temperature

was examined and it was found that temperature had a little

influence on the results in the temperature range of 15 to

P. H. YANG ET AL.489

0 102030405

F

t/min

Figure 7. The effect of incubating time on the process of

FRET at 25°C and 0.01 M PBS, and pH value was 7.4. The

anti-BSA–FITC concentration was 0.12 mg/mL, and the

GNPs concentration was 5.0 × 10–8 M.

3.0 4.5 6.0 7.5 9.010.512.0

-10

F

Figure 8. The effect of pH value on the process of FRET at

25°C and 0.01 M PBS, after 25 min of incubation. The an-

ti-BSA–FITC concentration was 0.12 mg/mL, and the GNPs

concentration was 5.0 × 10–8 M.

40°C. So, room temperature (25°C) was chosen for fol-

lowing work. The influence of the buffer systems and the

pH value on FRET was examined. The results showed pH

7.4 PBS buffer was the most suitable (Figure 8 ), which can

avoid antigen or antibody denaturation when solutions at

higher pH value or at lower pH value.

Under the above optimized conditions, the fluores-

cence quenching intensity is linearly proportional to BSA

concentration from 2.9 nM to 43.5 nM with a detection

limit of 0.5 nM, the linear regression equation was ∆F =

2.887 × CBSA + 7.007, with a correlation coefficient(R)

of 0.9935.

The serum was diluted about 100 times during the dis-

posal procedure (sample 1), the recovery test was done

by the standard addition method (n = 3). The recovery

values with their standard deviation are shown in Table

2. The average quenching intensity was 6.0%, which was

obtained from 10 replicate determinations of 4.3 × 10–8

M GNPs–BSA under the optimized conditions. The me-

thod recovery was calculated compared with three

known concentration levels. The recovery values are

shown in Table 2 (sample 2). The above results indi-

cated that the proposed method has a high repeatability

and precision.

4. Conclusions

We have demonstrated an indirect immunoassay based

on fluorescence resonance energy transfer. Antigen was

conjugated gold nanoparticles as acceptor, and FITC was

attached the corresponding antibody as FRET donor.

BSA and anti-BSA antibody were chosen as a model

antibody-antigen system. The formation of anti-BSA–

BSA immunocomplex resulted in the FRET between

donor and acceptor. The concentration of BSA is deter-

mined by quenching of fluorescently FRET acceptor.

This method has been successfully applied to determina

Table 2. Analytical results of serum samples and recovery.

samples Added (nM) Founda(nm) Recovery Recovery (mean)

0 33.50 ± 0.60 __

5.80 38.70 ± 0.54 97.67

20.30 57.10 ± 0.69 116.5

Sample 1

37.70 68.70 ± 0.52 92.45

102.2

5.80 6.07 ± 0.25 104.7

20.30 21.70 ± 0.91 106.9 Sample 2

37.70 35.90 ± 1.51 95.21

103.3

P. H. YANG ET AL.

490

tion of total serum protein in serum samples.

5. Acknowledgements

This project was supported by the Major State Basic Re-

search Development Program of China (973 Program)

(No. 2010CB833603) and the National Natural Science

Foundation of China (No. 21071064).

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