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Fluorescence Enhancement of Silver Nanoparticle Hybrid Probes and Ultrasensitive Detection of IgE Hui Li,† Weibing Qiang,† Maika Vuki,‡ Danke Xu,†,* and Hong-Yuan Chen† †
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, People's Republic of China ABSTRACT: An ultrasensitive protein assay method was developed based on silver nanoparticle (AgNP) hybrid probes and metal-enhanced fluorescence. Two aptamer based silver nanoparticles, Aptamer/Oligomer-A/Cy3-modified AgNPs (Tag-A) and Aptamer/Oligomer-B/Cy3-modified AgNPs (Tag-B) were hybridized to form a silver nanoparticle aggregate that produced a red shift and broadening of the Localized Surface Plasmon Resonance (LSPR) peak. The enhanced fluorescence resulted from the increased content of Cy3 molecules and their emission resonance coupled to the broadened localized surface plasmon (LSP) of AgNP aggregate. The separation distance between Cy3 and AgNPs was 8 nm which was the most optimal for metal enhanced fluorescence and the separation distance between adjacent AgNPs was about 16 nm and this was controlled by the lengths of oligomer-A and oligomer-B. The protein array was prepared by covalently immobilizing capture antibodies on aldehyde-coated slide. After addition of protein IgE sample, two kinds of aptamer-modified AgNPs (Tag-A and Tag-B) were employed to specifically recognize IgE and form the AgNP aggregate on the arrays based on their hybridization. The detection property of the aptamermodified AgNP aggregate was compared to two other modified aptamer-based probes, aptamer-modified Cy3 and Tag-A. The modified AgNP hybrid probe (Tag-A and Tag-B) showed remarkable superiority in both sensitivity and detection limit due to the formed AgNP aggregate. The new hybrid probe also produced a wider linear range from 0.49 to 1000 ng/mL with the detection limit reduced to 40 pg/mL (211 fM). The presented method showed that the newly designed strategy of combining aptamer-based nanomaterials to form aggregates results in a highly sensitive optical detection method based on localized surface plasmon.
’ INTRODUCTION The fluorescence response can be enhanced when the fluorophore is localized near a metal surface, which is defined as metalenhanced fluorescence (MEF).1,2 The MEF is related to the shape and size of the metal particle, and the distance between the dye and the metal surface.3,4 Metallic nanoparticle and nanostructures have excellent optical properties due to excitation of their localized surface plasmons by incident light, which results in strong surface plasmon absorption bands and an enhancement of the local electromagnetic fields.5,6 The spectral overlap between the fluorophore emission and the plasmonic resonance results in fluorescence enhancement.79 Farcaua7 reported an emission enhancement of 28 times for Rose Bengal fluorophore when placed at about 1 nm above silver half-shells. Tam8 also reported an enhancement factor of 50 when plasmon resonance of Au nanoshells overlapped with the indocyanine green (ICG) emission wavelength whereas Chen9 found that the overlap between the LSPR and the emission spectra is a critical factor for fluorescence enhancement and estimated the absolute fluorescence enhancement factor to be 930 for the brightest individual particles. Since the fluorescence intensity of a dye within the proximity of a nanoparticle is strongly dependent on the overlap between the LSPR of the nanoparticle and the spectral properties r 2011 American Chemical Society
of the dye, controlling the modulation of the LSPR of nanoparticle is extremely important for fluorescence enhancement. Silver Nanoparticles (AgNPs) have been widely used for MEF studies1014 and the optimal distance between the dye and metal for the largest fluorescence enhancement was reported to be 8 nm.15,16 The greater MEF effect has also been observed when the fluorophores are simultaneously coupled to two nanoparticles, the hybridization between AgNPs will cause a red shift and broadening of LSPR peak of nanoparticles17 that results in a strong metal-enhanced fluorescence.15 Zhang15 using single Cy5 molecule modified AgNPs on hybridized metal monomer and metal dimer found the metal dimer to have a stronger fluorescence enhancement, 13-fold, compared to the free Cy5-labeled oligonucleotide. The LSPR of the metal dimer provided a better overlap with the emission of Cy5 resulting in the enhanced fluorescence response. The limitation in using the metal fluorescent monomer or dimer as detection tags in biochemical analysis may due to the complications ensued in the multiple functionalization of silver nanoparticles. We have reported using Received: June 21, 2011 Accepted: October 10, 2011 Published: October 10, 2011 8945
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Scheme 1. Schematic Illustration of Procedure of Immunoassay and Detection Strategy by Sandwich-Type with Hybrid Probes
silver nanoparticles and its aggregate as detection tags in electrochemical detection for DNA18 and also developed a successful protocol in the modification of silver nanoparticle with several kinds of oligonucleotides and the formation of aggregate. In this work, we report on modified AgNPs with multifunctional oligonucleotides, in which the DNA aptamer was used as recognition molecule, fluorescent dyes as detection molecule, Oligomer-A and Oligomer-B as hybridization molecules to form aggregate resulting in secondary fluorescence enhancement. Aptamers are DNA/RNA oligonucleotides selected from combinational oligonucleotide libraries by systematic evolution of ligands by exponential enrichment (SELEX).19,20 Aptamer has a defined three-dimensional shape that allows them to interact, with high affinity, with a target molecule. Aptamers are easily synthesized or modified, and are chemically stable.21 IgE, chosen as target molecule, plays a very important role in allergic reactions and other related diseases in human and it is critical to establish a simple and ultrasensitive method for analyzing IgE. A welldesigned DNA aptamer specific for IgE22 was selected as recognition molecule to substitute for antibody. Aptamer-based methods have been widely reported, such as label-free surface plasmon resonance spectroscopy (SPR),23,24 quartz crystal microbalance (QCM),25 capillary electrophoresis (CE),26 electrochemical method,2730 and fluorescence method.31,32 Recently, aptamers have been used to couple with nanoparticles to develop higher sensitive labels for human IgE determination.33 Although silver enhanced fluorescence enhancement had been widely studied1014 and used in protein assays,34 to our best knowledge, silver nanoparticles functionalized both with fluorescent molecules and aptamers have not been reported to assay protein. In this work, we developed an ultrasensitive fluorescent detection method for protein IgE by combining the aptamer that specifically recognizes IgE with the AgNP hybrid probes. Silver nanoparticle aggregates were formed in situ on the arrays by the hybridization of Aptamer/Oligomer-A/Cy3-modified AgNPs (Tag-A) and Aptamer/Oligomer-B/Cy3-modified AgNPs (Tag-B), and the strategy of detection is shown in Scheme 1. Compared to
our electrochemical method reported previously, the presented method eliminates the step for the synthesis of aggregate tags prior to immobilization. The novel hybridized-induced Tag was successfully applied to combine with target protein IgE by sandwich format and the fluorescent intensity of the aggregate formed by the hybridization showed greater enhancement due to the increased Cy3 modified AgNPs and MEF effect.
’ EXPERIMENTAL SECTION Materials and Reagents. Aldehyde slide (Shanghai Biotechnology Co. Ltd.), Silver nitrate, and sodium borohydride (Sigma Aldrich) were used to synthesize colloidal silver nanoparticles; anti human IgE produced in goat (Sigma Aldrich), IgE kappa, Myeloma, Human (United States Biological, USA) were proteins for target and detection; Phosphate buffered saline (PBS) (Shanghai Sengon Biotechnology Co.), tween-20 (Nanjing Bokkman Biotechnology Ltd.), NaCl (Nanjing Chemical Reagent Co. Ltd.) were used for preparation of the following phosphate buffers: 1 PBS (137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L, Na2HPO4.12H2O, 2 mmol/L KH2PO4, pH 7.4), 0.1 M PBS (0.1 M NaCl + 0.1 PBS), 0.1 M PBST (0.1 MPBS + 0.05% tween-20), 0.2 M PBS+ (0.2 M NaCl + 0.1 PBS + 1 mM MgCl2). Doubly distilled deionized water was used in all experiments. All of the synthetic oligonucleotides used in this study were purchased from Shanghai Sengon Biotechnology Co. The oligonucleotides were derived with alkyl thiol group at the 50 terminus and their sequences are as follows: 50 SH-oligo(d)A24-Cy3: 50 SH-AAAAAAAAAAAAAAAAAAAAAAAA-Cy3, 50 SH-oligo(d)A15: 50 SH-AAAAAAAAAAAAAAA, Aptamer: 50 SH-AAAAAAAAAAAAAAAGGGGCACGTTTATCCGTCCCTCCTAGTGGCGTGCCCC, Aptamer-modified Cy3: GGGGCACGTTTATCCGTCCCTCCTAGTGGCGTGCCCCAAA-Cy3, Oligomer-A: 50 SH-AAAAAAAAAAAAAAAGGGTGCTCCCCCTAGTGG, 8946
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Analytical Chemistry Oligomer-B: 50 SH-AAAAAAAAAAAAAAACCACTAGGGGGAGCACCC, Apparatus. The absorption for LSPR spectra were measured on UVvis spectrophotometer (Shimadzu UV3600, Japan), fluorescence spectra of 50 SH-oligo(d)A24-Cy3 were collected on a spectrofluorometer (RF-5301PC, Shimadzu, Japan). The immunological arrays were made by Smart Arrayer (Beijing CapitalBio Co. Ltd., China) and scanned by Luxscan-10K/A microarray Scanner (532 nm laser source for Cy3, Beijing CapitalBio Co. Ltd., China). Scanning electron microscopy (SEM) (S-4800, Japan) and Transmission electron microscope (TEM) (JEM-200CX, Japan) were used for collecting SEM and TEM images and CT15RT versatile refrigerated centrifuge from TECHCOMP (Shanghai, China). Preparation of Silver Nanoparticles. Silver nanoparticles were prepared using our published method.18 Briefly, ice cold AgNO3 (2 103 M) was added dropwise in twice the volume of NaBH4 (3 103 M) with vigorous stirring in an ice bath. After the addition of all AgNO3, 8 mL NaBH4 was added to make the solution become bright yellow in a hot bath. Finally, the solution was vigorously stirred to room temperature and stored at 4 °C. Preparation of Tag-A. The modification of silver nanoparticle by SH-oligo(d)A24-Cy3, SH-oligo(d)A15, Oligomer-A, and Aptamer were prepared according to the previously published method with some modifications.18 Briefly, 1 mL of silver nanoparticle solution mixed with SH-oligo(d)A24-Cy3 (25 μL, 10 μM), SH-oligo(d)A15 (25 μL, 10 μM), Oligomer-A (25 μL, 10 μM), and Aptamer (25 μL, 10 μM) were incubated for 18 h. 122 μL of 1 PBS was added to the solution and allowed to react for 6 h. Then 21 μL of 2 M NaCl was added to the solution and this step was repeated after a 3 h interval. A 26 μL portion of 2 M NaCl was then added to the solution after 12 h and again this step was repeated after a 3 h interval to allow the total NaCl concentration to increase gradually to 0.2 M. After an additional standing of at least 48 h, the modified nanoparticles were isolated by centrifugation for 15 min, 14 °C, 3 times at 15 000 rpm. The resulting precipitate was washed and recentrifuged in 0.1 M PBS and redispersed in 1368 μL of 0.2 M PBS+. Preparation of Tag-B. The modification of silver nanoparticle by SH-oligo(d)A24-Cy3, SH-oligo(d)A15, Oligomer-B, and Aptamer follows the same procedure as for Tag-A. Briefly, 1 mL aliquots of silver nanoparticle solution were mixed with SH-oligo(d)A24-Cy3 (25 μL, 10 μM), SH-oligo(d)-A15 (25 μL, 10 μM), Oligomer-B (25 μL, 10 μM), and Aptamer (25 μL, 10 μM) and incubated for at least 18 h. The rest of the steps were the same as above. Preparation of Tag-C. The modification of silver nanoparticle by SH-oligo(d)A15, Oligomer-B, and Aptamer follows the same procedure as for Tag-B. One milliliter aliquots of silver nanoparticle solution were mixed with SH-oligo(d)-A15 (50 μL, 10 μM), Oligomer-B (25 μL, 10 μM), and Aptamer (25 μL, 10 μM) and incubated for at least 18 h. The rest of the steps were the same as above. Analytical Procedure. A 4 4 microarray was fabricated on aldehyde-modified slides (3 6 = 18 reaction pond) by Smart Arrayer. First, 0.1 mg/mL of anti human IgE was immobilized on the slide and left to stand for 4 h at 37 °C. Then the slide was blocked by reacting with buffer (10 mg/mL BSA) for 1 h and washed with 0.1 M PBST 3 times, at 5 min for each washing. Third, 30 μL of different concentrations of IgE were added and allowed to react for 1 h at 37 °C followed by the washing step as described before. Finally, the mixture of Tag-A (1.46 nM) and
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Tag-B (1.45 nM) was added to bind with IgE at 37 °C for 1 h and then subjected to the same washing step as before. The array was scanned and the data were collected by Luxscan-10K/A microarray Scanner. Metal Enhanced Fluorescence Identification. According to the analytical procedure, Tag-A was first reacted with IgE sample for 1 h, after washing step, followed by the addition of the different Tags (Tag-A, Tag-B, Tag-C) on the slides for 1 h and then subjected to the same washing step as before. The array was scanned and the data were collected by Luxscan-10K/A microarray Scanner. Linear Relationship and Detectability of Tags. For the determination of linear relationship, the Tags were added to IgE concentrations ranging from 7.8 to 1000 ng/mL with reaction time of 60 min at 37 °C. However, for concentrations below 7.8 ng/mL, the reaction time was reduced to 15 min. To establish the detection limit, the reaction time for IgE and Tags was also 15 min, otherwise the rest of steps were the same as in the Analytical procedure. LSPR Peak Measurements. A 600 μL portion of Tag-A or Tag-B was centrifuged and redispersed in 300 μL 0.2 M PBS+. Then Tag-A was mixed with Tag-B for 0, 15, 30, 60, and 120 min at 37 °C with gentle shaking. The LSPR spectra of Tag-A, Tag-B, and the resulting aggregate solution (50 μL Tags or the aggregate was diluted by 200 μL 0.2 M PBS+ and 100 μL silver nanoparticle was diluted by 150 μL doubly distilled deionized water) were measured at 0, 15, 30, 60, and 120 min by UVvis spectrophotometer (Shimadzu UV3600, Japan). Transmission Electron Microscope Images. A 600 μL portion of Tag-A or Tag-B was centrifuged and redispersed in 300 μL 0.2 M PBS+. Then Tag-A was mixed with Tag-B for 60 min at 37 °C with gentle shaking. Then, the solution was added on the carbon film for TEM images recording. Fluorescence Intensity Measurements. The fluorescence intensity were measured using a Luxscan-10K/A microarray Scanner (λex = 532 nm) and the emission spectra of Cy3 by a spectrofluorometer.
’ RESULTS AND DISCUSSION Property of Tag-A, Tag-B and AgNP Aggregates. The fabrication of the AgNP aggregate and analytical procedure of protein arrays by sandwich-type is shown in Scheme 1. The attachment of SH-oligo(d)A24-Cy3, SH- oligo(d)A15, Oligomer-A, Oligomer-B, and the Aptamer to AgNPs were attained by bonding through the sulfhydryl group (SH) at 50 terminus. The polyadenine (A) was used to stabilize the AgNPs and increase the yield of modification of Tag-A and Tag-B. Both Tag-A and Tag-B have similar properties. First, as shown in Figure 1(a,b), they have the similar LSPR peak which imply that they have similar metal enhance fluorescence effect. Second, they have the similar size and morphology and most silver nanoparticles are spherical as shown in Figure 2(A,B), the diameter of Tag-A is 20.0 ( 4.2 nm (N = 102) and Tag-B is 19.3 ( 4.7 nm (N = 122). As TagA and Tag-B have similar properties, we choose Tag-A as detection Tag in our experiment to compare with aptamer modified Cy3 and the hybrid probes. However, the property of the hybrid of Tag-A and Tag-B is totally different from the individual Tag-A or Tag-B. The LSPR peaks of AgNP aggregate are broader and the absorbance was lower than Tag-A and Tag-B (Figure 1A). Tag-A and AgNP aggregate are not only different in LSPR peak but also in size and morphology (see Figure 2). 8947
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Figure 1. (A) The LSPR peak of Tags, a: Tag-A(0.58 nM), b: TagB(0.58 nM), c: 0 min (2.92 nM Tag-A mix with 2.90 nM Tag-B in solution at 37 °C with gentle shaking, then 50 μL hybrid probes was diluted by 200 μL 0.2 M PBS), d: 15 min, e: 30 min, f: 60 min, g: 120 min, h: Emission spectra of Cy3 at 532 nm excitation wavelength, I: the absorption of AgNPs(100 μL AgNPs was diluted by 150 μL doubly distilled deionized water). (B) (1) the fluorescence intensity ratio of Tag-B and Tag-A(F[Tag‑B+Tag‑A]/F[Tag‑A+Tag‑A]), (2) the fluorescence intensity ratio of Tag-C and Tag-A(F[Tag‑C+Tag‑A]/F[Tag‑A+Tag‑A]).
This type of silver aggregate was synthesized and characterized in our previous article where the size and micrograph of the aggregate was controlled by diluting the aggregate.18 In this method, AgNP aggregate could be prepared in situ on the array rather than using a prepared AgNP aggregate as fluorescent Tag. To observe fluorescence property as well as the size and micrograph of AgNP aggregate, however, the AgNP aggregate was also prepared in the solution by the hybridization and its size was controlled by the incubation time, using the same conditions as the hybridization between Tag-A and Tag-B on the slides. The experimental results showed that the resulting AgNP aggregate had a good homogeneity as shown in Figure 2(CF). TEM coupled with SEM images indicate that the sizes of the modified AgNP aggregate are about 450 nm and the morphology is like round. On the basis of the previous work,35 the concentrations of AgNPs were calculated as 2.60 and 2.80 nM, respectively for Tag-A and Tag-B. On the basis of Figure 1(i,a,b), the concentrations of Tag-A and Tag-B were 1.46 and 1.45nM, respectively. The concentrations of SH- oligo(d)-A24-Cy3 on the Tag-A and Tag-B were calculated as 31.72 pmol and 30.38 pmol, respectively. These correspond to 22 SH-oligo(d)-A24-Cy3 units and 21 SHoligo(d)-A24-Cy3 units on the surface of Tag-A and Tag-B respectively. If four kinds of oligonucleotide have the same opportunity to couple with AgNPs, then there would be 88 oligonucleotide units per particle for Tag-A, and 84 oligonucleotide
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units per particle for Tag-B. Theoretically, the fluorescence intensity of Tag-A should be more than 22 times higher than that of aptamer-modified Cy3. However, this fluorescence enhancement might occur in theoretical solution environment rather than in biomolecular immobilization interfaces such as the arrays due to stereo hinder of nanoparticles and angle of incident light. As a result, in our experiment, an amplification of 12 times was obtained from Tag-A. The hybridization resulted in aggregation of Tag-A and Tag-B into a larger hybrid modified AgNP with a simultaneous change in the localized surface Plasmon of the AgNPs as was observed from the UVvis spectroscopy (Figure 1A). The LSPR peak of AgNP aggregate showed a red shift of ∼2.5 nm (from 403.5 nm to 406 nm) and slight broadening that occurred at different incubation times from 15 to 120 min. The shift and the broadening could both be attributed for the fluorescence enhancement. The absorbance of AgNP aggregate decreased from 0.58 to 0.24 at 403.5 nm, which reflects that only a portion of Tag-A and Tag-B was hybridized with each other. The absorbance of AgNPs can also reflect the concentration of Tag-A and Tag-B. A decrease in absorbance could indicate a decrease in the number of Tag-A and Tag-B and we found that only 55% Tag-A and 52% Tag-B are combined to form the aggregate. This is calculated using the ratio of absorbance at 403.5 nm from Figure.1 and the fact that 45% tag-A and 48% Tag-B are still in single nanoparticle. The absorbance of AgNP aggregate increased from 0.014 to 0.050 at 562 nm which corresponded to the maximum emission wavelength of Cy3. The broadening of the AgNP aggregate LSPR peak provided a better overlap with the emission spectra of Cy3 which resulted in the greater fluorescence enhancement. The broadening of AgNP aggregate LSPR peak implies that more silver nanoparticles hybridize with each other and the number of Cy3 molecule increase. The sandwich assay hybridization between oligonucleotide modified AgNPs and OligonucleotideCy3 have been reported for metal enhanced fluorescence and that a metal dimer complex had a stronger fluorescence enhancement than a single silver nanoparticle mononer.15 The increased enhancement based on formation of AgNP aggregate from this study clearly supports the observed enhancement effect for metal dimer complex. However, the application of AgNP aggregate for fluorescence enhancement has never been reported as detection tags. We identified the metal enhanced fluorescence of AgNP aggregation and the result is shown in Figure1(B). The fluorescent intensity ratio of Tag-B and Tag-A was found to be 9.1(F[Tag‑B+Tag‑A]/F[Tag‑A+Tag‑A] = 9.1), and Tag-C and Tag-A is 2.4(F[Tag‑C+Tag‑A]/F[Tag‑A+Tag‑A] = 2.4). The lower fluorescent intensity ratio of Tag-C and Tag-A could be attributed to the fluorescent enhancement of MEF only, whereas the higher fluorescence intensity ratio for Tag-B and Tag-A could be due to the combined fluorescence enhancement of MEF and increase in Cy3 content. Therefore, we conclude that the fluorescence signal is enhanced by MEF of AgNP aggregation and the increase in Cy3 content in the aggregate complex. The stability of AgNPs prepared has also been studied. As oxidization will change the LSPR peak of AgNPs, UV absorbance of AgNPs was measured after the desired storing time. Actually, our experimental results showed that the UV absorbance of AgNPs could be kept constant after storing at 4 °C for 57 days and the relative LSPR peak show no obvious changes. Thus, the AgNPs could be kept stable at longer times under the stated conditions. 8948
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Figure 2. TEM of Tag-A(A),TEM of Tag-B(B),TEM of hybrid probes formed by Tag-A and Tag-B reacting for 1 h in solution and then the solution was added on the carbon film (C,D). SEM of hybrid probes formed by Tag-A and Tag-B reacting for 1 h on Aldehyde Slide accroding to the analytical procedure (E,F).
Figure 3. (A) The fluorescence intensity of Signal and background of BSA, the concentration of IgE and BSA were 167 and 1 mg/mL (a), the insert curve b is the relationship of time and S/N,1:IgE, 2:BSA, (B) The relationship of Fluorescence Intensity Ratio and the incubation time, 1:F[Tag‑A]/ F[aptamer modified Cy3], 2: F[hybrid probes]/F[aptamer modified Cy3] at 167 ng/mL IgE. (C) the relationship of Fluorescence Intensity Ratio and IgE concentration at 1 h incubation time, 1:F[Tag‑A]/F[aptamer modified Cy3], 2: F[hybrid probes]/F[aptamer modified Cy3].
Fluorescence Enhancement of AgNP Hybrid Probes. To determine the level of fluorescence enhancement of AgNP hybrid probes, the fluorescent intensities were assayed as a function of hybridization time of Tag-A with Tag-B and the results were shown in Figure 3A(a). With the increase of incubation time, a remarkable increase in the fluorescence intensity of hybrid probes was also obtained. However, the fluorescence intensity of BSA, the control protein, also increased but with much lower intensity, and the fluorescence intensity ratio of Signal and BSA (divide the fluorescence intensity of Signal by BSA, Fsignal/FBSA = S/N) was highest at 60 min, as shown in Figure 3A(b). The results indicated that the hybrid probe of Tag-A and Tag-B could effectively form the AgNP aggregates in situ on the protein arrays. In addition, there was no buffer when the aggregation was scanned, the thermal denaturation will not affect the experimental results.
To prove fluorescence enhancement phenomenon due to the formed AgNP aggregate, two other Cy3 labeled aptamers (aptamer modified Cy3 and Tag-A) were used to compare the fluorescent intensities. According to Figure 3(B) that the fluorescence intensity ratio of the hybrid probes to the aptamer modified Cy3 increased from 36-fold to 126-fold when the incubation time was increased from 15 min to 120 min, while the fluorescence intensity ratio of Tag-A to the aptamer modified Cy3 remained almost constant of 12-fold. Figure 3(C) shows their fluorescence intensity ratios of the hybrid and Tag-A probes to the free aptamer modified Cy3 at the different IgE concentrations. The results clearly showed that the fluorescence intensity ratios of the hybrid probes to the aptamer modified Cy3 increased with the decrease of IgE concentration, while the ratio of Tag-A to the aptamer modified Cy3 slightly decreased. These results suggest that the hybrid probes gave more superior fluorescent 8949
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Figure 4. The linear relationship of aptamer-modified Cy3(A), TagA(B), and hybrid probes(C,D) between fluorescence intensity and logC (C means IgE concentration) and the fluorescence image of aptamer-modified Cy3, Tag-A, and hybrid probes at different IgE concentrations (E).
enhancement than the method using single AgNPs strand (Tag-A), especially in low concentration range of proteins. This conclusion may result from the steric hindrance of the Tags. The steric hindrance of hybrid probes in high IgE concentration is stronger than that in low IgE concentration. Therefore, the fluorescence intensity ratio of hybrid probes and Tag-A are larger at low IgE concentration. Development of Fluorescence Detection Strategy. Figure 4(AD) shows a calibration curve for aptamer modified Cy3, Tag-A, and hybrid probes. As for aptamer-modified Cy3, a good linear relationship between the fluorescence intensity (subtracting the intensity of BSA) and the logarithm values of IgE concentrations ranging from 31.2 ng/mL to 1000 ng/mL
(Y1 = 221.7X-318.4) and the correlation coefficients was 0.996. Tag-A and hybrid probes also show good linear relationship between the fluorescence intensity and the logarithm values of IgE concentration ranging from 7.8 to 1000 ng/mL (Y2 = 903.6X-762.9, Y3 = 4489X-3283) and the correlation coefficients were 0.983 and 0.990 respectively. In addition, hybrid probes also show a good linear relationship in the lower concentration range, from 0.497.8 ng/mL (Y4 = 216.5X-439.5), and the correlation coefficient was 0.997. The corresponding fluorescence images of aptamer modified Cy3, Tag-A, and hybrid probes at different IgE concentrations are also shown in Figure 4E. The limit of detection was determined with 1 μg/mL IgG used as a control and 15 min for incubation time to ensure a lower background. 8950
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Figure 5. The fluorescence intensity of ultralow concentration IgE using aptamer modified Cy3(A), TagA(B), and hybrid probes (C).
’ CONCLUSIONS We have demonstrated here a simple, sensitive, specific, and cost-effective method for quantitative determination of proteins. The hybrid AgNP aggregate used in this new method is superior to a single-strand AgNP or free aptamer labeled probes in the detection of IgE. Compared to the previously reported fluorescent methods, our aggregate-enhanced fluorescence method showed greater sensitivity and specificity in detection of proteins such as IgE. The novel fluorescent method reduced the LOD by 390 times and gave a wider linear range with good reproducibility, accuracy, and sensitivity. The convenient, ultrahigh sensitivity, and cost-effective approach of the silver aggregate enhanced fluorescence method provides great promise in protein analysis, particularly for ultralow level detection. ’ AUTHOR INFORMATION Figure 6. The repeatability of different reaction pond for hybrid probes, a: the intensity of curve b (1 + 10%), b: the intensity of the average of all samples from 18 incubation pond, and c: the intensity of curve b (110%).
Corresponding Author
*Tel/Fax: (+)00862583595835; E-mail:
[email protected]. Present Addresses ‡
The detection limit for aptamer modified Cy3 was 15.6 ng/mL for Tag-A was 1 ng/mL (5.10 pM) and hybrid probes was 0.04 ng/mL (211 fM) at signal-to-noise ratio of 3 (detection limit ≈ FIgG + 3SD) respectively. The measured results for ultralow concentration level detection are as shown in Figure 5.36 The detection limit of hybrid probes was 0.04 ng/mL corresponds to 6.33 amol of the target in the 30 μL sample solution, which is 390 times lower than aptamermodified Cy3. The detection limits obtained for Tag-A and hybrid probes compares favorably with the other optical methods summarized by Peng.37 For the fluorescence method, the lowest detection limit reported was 10 pM38 which is higher than the detection limit of hybrid probes obtained in this study (211fM). The reproducibility test was accomplished in different incubation ponds on a piece of aldehyde-modified slide. Using the optimal condition and the IgE concentration of 100 ng/mL, the reproducibility response is as shown in Figure 6. Among the 18 incubation ponds, 15 incubation ponds have intensity values within the 90110% intensity with RSD (Relative Standard Deviation) = 5.73%. These results demonstrated that hybrid probes have good reproducibility and precision, and therefore have great potential for application.
College of Natural and Applied Sciences, University of Guam, Mangilao, Guam 96923, United States of America.
’ ACKNOWLEDGMENT We acknowledge the financial support of the National Basic Research Program of China (973 Program, 2011CB911003), National Natural Foundation of China (Grant Nos. 20975050 and 21175066), The National Science Funds for Creative Research Groups(NO.20821063) and National Basic Research Program of China (973 program, No. 2007CB936404). M.V. acknowledges the support of the University of Guam. ’ REFERENCES (1) Aslan, K.; Gryczynski, I.; Malicka, J.; Matveeva, E.; Lakowicz, J. R.; Geddes, C. D. Curr. Opin. Biotech. 2005, 16, 55–62. (2) Hong, G. S.; Tabakman, S. M.; Welsher, K.; Wang, H. L.; Wang, X. R.; Dai, H. J. J. Am. Chem. Soc. 2010, 132, 15920–15923. (3) Chan, Y. H.; Chen, J. X.; Wark, S. E.; Skiles, S. L.; Son, D. H.; Batteas, J. D. ACS Nano 2009, 3 (7), 1735–1744. (4) Cheng, D. M.; Xu, Q. H. Chem. Commun. 2007, 248–250. (5) Hutter, E.; Fendler, J. H. Adv. Mater. 2004, 16 (9), 1685–1705. (6) Sepulvedaa, B.; Angelomeb, P. C.; Lechugaa, L. M.; Liz-Marzan, L. M. Nano Today 2009, 4, 244–251. (7) Farcaua, C.; As-tilean, S. Appl. Phys. Lett. 2009, 95, 193110. 8951
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Analytical Chemistry
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dx.doi.org/10.1021/ac201574s |Anal. Chem. 2011, 83, 8945–8952
