Silver Nanoparticle-Enhanced Fluorescence Resonance Energy

Mar 26, 2013 - A silver nanoparticle (AgNP)-enhanced fluorescence resonance ..... Aptamer and rolling circle amplification-involved sandwich assay for...
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Silver Nanoparticle-Enhanced Fluorescence Resonance Energy Transfer Sensor for Human Platelet-Derived Growth Factor-BB Detection Hui Li, Min Wang, Chongzhi Wang, Wei Li, Weibing Qiang, and Danke Xu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, China ABSTRACT: A silver nanoparticle (AgNP)-enhanced fluorescence resonance energy transfer (FRET) sensing system is designed for the sensitive detection of human platelet-derived growth factor-BB (PDGF-BB). Fluorophore-functionalized aptamers and quencher-carrying strands hybridized in duplex are coupled with streptavidin (SA)-functionalized nanoparticles to form a AgNP-enhanced FRET sensor. The resulting sensor shows lower background fluorescence intensity in the duplex state due to the FRET effect between fluorophores and quenchers. Upon the addition of PDGF-BB, the quencher-carrying strands (BHQ-2) of the duplex are displaced leading to the disruption of the FRET effect. As a result, the fluorescent intensity of the fluorophore−aptamer within the proximity of the AgNP is increased. When compared to the gold nanoparticle (AuNP)-based FRET and bare FRET sensors, the AgNP-based FRET sensor showed remarkable increase in fluorescence intensity, target specificity, and sensitivity. Results also show versatility of the AgNP in the enhancement of sensitivity and selectivity of the FRET sensor. In addition, a good linear response was obtained when the PDGF-BB concentrations are in the ranges of 100−500 and 6.2−50 ng/mL with the detection limit of 0.8 ng/mL.

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very limited progress in the development of bioanalytical methods for the detection of proteins using this approach. In this study, a nanoparticle-based FRET sensor was designed using fluorophore-functionalized aptamers that are hybridized with quencher BHQ-2 carrying strands for the detection of platelet-derived growth factor-BB (PDGF-BB). Three factors pertaining to FRET sensing were considered. First is the suitable distance between silver nanoparticles and the FRET system (a donor and acceptor). Second is the appropriate interaction between a donor and acceptor, and third is the functionalization of silver nanoparticles with biological molecules. Aptamers are short, single-stranded oligonucleotides generated from an in vitro method known as SELEX (systematic evolution of ligands by exponential enrichment).27 Compared to antibodies, aptamers have unique advantages such as easy synthesis, good stability, and chemical modification.28,29 Aptamer-based affinity methods have attracted more and more attention as an alternative to antibody.30,31 Furthermore, the length of aptamer can be regulated by connecting a spacer at one end of the binding target. The length of the aptamer could also be used for controlling the distance between

luorescence resonance energy transfer (FRET) involves the nanoradiative transfer of excitation energy from an excited state donor to a proximal ground state acceptor.1,2 FRET-based sensors have been widely used in bioassay sensors,3−5 such as metal ion detection,6,7 DNA,8 and cell,9 where the sensing systems have been designed in the form of a donor−acceptor system which contains a fluorophore and a quencher. For example, fluorophore−fluorophore systems have been used as donor−acceptor for FRET sensing systems.10 Recently, more novel materials such as quantum dots, gold nanoparticles, silica, and polymer particles have been reported for the development of FRET sensors. Quantum dots have fluorescent properties and can also be a donor or acceptor in FRET systems,11−13 whereas gold nanoparticles are regularly used as acceptors to quench the fluorescence response of the fluorophores.14−17 Silica18,19 and polymer particles20−22 have been used as shells to incorporate fluorescent dyes to form fluorescent nanoparticles that make the FRET systems water dispersible as well as to enhance fluorescent brightness and photostability. On the other hand, silver nanoparticles could enhance the FRET efficiency through silver-fluorophore interaction, leading to an increase in quantum yield and emission intensity of the fluorophore.23−25 Holmes-Smith26 showed that the silver nanostructures enhance the efficiency of FRET between donor molecule (Rhodanmine 6G) and acceptor molecule (Texas Red). Although the phenomenon of silver-enhanced FRET efficiency has been reported, there is © 2013 American Chemical Society

Received: January 7, 2013 Accepted: March 26, 2013 Published: March 26, 2013 4492

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fluorescent dyes and silver nanoparticles in order to establish a silver-based FRET sensor with excellent performance. In this study, we report on a silver nanoparticle (AgNP)based FRET sensor for the sensitive detection of human PDGF-BB (Scheme 1). Fluorophore-functionalized aptamers

Apt-59bp: 5′TAMRA-CAGGCTACGGCACGTAGAGCATCACCATGATCCTGAAAAAAAAAAAAAAAAAAAAAAAA-Biotin-3′, TAMRA (Carboxytetramethylrhodamine) Apt-53bp: 5′TAMRA-CAGGCTACGGCACGTAGAGCATCACCATGATCCTGAAAAAAAAAAAAAAAAAA-Biotin3′ Apt-47bp: 5′TAMRA-CAGGCTACGGCACGTAGAGCATCACCATGATCCTGAAAAAAAAAAAA-Biotin-3′ BHQ-2(15bp): 5′-ACGTGCCGTAGCCTG-BHQ-2-3′, BHQ-2(Black hole quencher-2) BHQ-2(12bp): 5′-TGCCGTAGCCTG-BHQ-2-3′ BHQ-2(9bp): 5′-CGTAGCCTG-BHQ-2-3′ Apparatus. The ultraviolet−visible (UV−vis) spectra and fluorescence intensity (excitation wavelength was λex = 532 nm and emission wavelength was λem = 582 nm) were measured on BioTek (Synergy H1, USA); a transmission electron microscope (TEM) (JEM-200CX, Japan) was used for collecting TEM images. Preparation of Silver Nanoparticles. Silver nanoparticle (AgNP-1) was synthesized according to the published method with some modification,34 and classical silver nanoparticle (AgNP-2) was prepared using our published method.35,36 Five milliters AgNP-2 was mixed with 5 mL of polyvinylpyrrolidone (PVP, 1%) and 5 mL of 10 mM sodium L-ascorbate for 5 min. Then, 5 mL of 10 mM AgNO3 was added and stirred for 1 h to prepare AgNP-1. Preparation of Gold Nanoparticles. Gold nanoparticle (AuNP) was prepared according to the procedure described in a previous article with some modification.37 Briefly, sodium citrate (2.2 mmol/L, 150 mL) was heated under reflux with an oil bath for 15 min with vigorous stirring in a 250 mL threenecked round-bottomed flask to prevent the evaporation of the solvent. Then, HAuCl4 (25 mmol/L, 1 mL) was quickly injected to the above boiling solution. The solution turned from yellow to bluish gray and then to soft pink within 10 min. Then, the mixture was cooled to 90 °C, and HAuCl4 solution (25 mmol/L, 1 mL) was again injected. After 30 min, heating was removed and the prepared AuNPs solution was cooled to room temperature and was stored at 4 °C for further use. SA-Functionalized Nanoparticles. One milliliter of AgNP-1 was mixed with 200 μL of 1 mg/mL SA for 3 h with gentle shaking at 37 °C. The the mixture was centrifuged at 15 000 rpm for 15 min, washed, and then redispersed with 1 mg/mL BSA to make SA-functionalized AgNP-1. In the same way, 1 mL of AgNP-2 or AuNP was mixed with SA to make SAfunctionalized AgNP-2 and AuNP. Fabrication of FRET Sensor. One milliliter of SAfunctionalized AgNP-1 was mixed with 200 μL of 1 μM Apt53bp and 200 μL of 2 μM BHQ-2 (12bp) for 1 h with gentle shaking at 37 °C; then, the mixture was centrifuged at 15 000 rpm for 15 min. The mixture was washed and redispersed with 1× PBSM as before. Strategy for Detection of PDGF-BB. Different concentrations of PDGF-BB in the range of 0.8−50 ng/mL (170 μL) were added to the FRET sensor (50 μL) for 70 min at 47 °C in a 96 black microwell plate; then, the plate was scanned using BioTek, and the data was collected. The excitation wavelength was 532 nm, and emission wavelength was 582 nm.

Scheme 1. Schematic Illustration of the Preparation of the Silver Nanoparticle-Enhanced FRET Sensor and the Determination of Human PDGF-BB

and quencher-carrying strands which were hybridized in duplex were bound with streptavidin (SA)-functionalized nanoparticles to form a AgNP-based FRET sensor. Human platelet-derived growth factor-BB (PDGF-BB) is an important cytokine in serum that serves as an indicator for tumor angiogenesis.32 The aptamer of PDGF-BB33 is used to specifically bind with PDGFBB. The FRET sensor shows lower fluorescence in the duplex sate. However, the addition of PDGF-BB results in the dispalcement of the quencher-carrying strands (BHQ-2), thus disrupting the FRET effect, and the appearance of silver enhanced fluorescence on fluorophores. The results show the AgNP-based FRET sensor is superior to bare FRET sensor and gold nanoparticle (AuNP)-based FRET sensor in detection of PDGF-BB. Silver nanoparticles can enhance the sensitivity of the FRET sensor, and the AgNP-enhanced FRET sensor has good selectivity. In addition, good linear response is found when PDGF-BB concentration is in the range of 100−500 and 6.2−50 ng/mL; the detection limit is determined as 0.8 ng/mL.

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METHODS Materials and Reagents. 96 microwell plates were utilized for quantifying fluorescence signals (Coring incorporated, NY, USA); silver nitrate (AgNO3), sodium borohydride (NaBH4), sodium L-ascorbate, and polyvinylpyrrolidone (PVP, MW = 40 000) were purchased from Sigma Aldrich (St, Louis, MO, USA). PDGF-BB (MW = 24 800 Da, R&D System, Minneapolis, MN), streptavidin, (SA, 58 kDa Beijing Biosynthesis biotechnology Co., LTD), and normal human serum (Zhongkechenyu Biotech, Beijing, China) were also used. Bovine serum albumin (BSA) was obtained from Amresco (Solon, USA); phosphate buffered saline (PBS) (Shanghai Sangon Biotechnology Co.), 1× PBS (137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L, Na2HPO4·12H2O, 2 mmol/L KH2PO4, pH = 7.4), 1× PBSM (1× PBS + 1 mmol/L MgCl2) was used. All the synthetic oligonucleotides used in this study were purchased from Shanghai Sangon Biotechnology Co. The sequences of oligonucleotides are as follows:

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RESULTS AND DISCUSSION Characterization of Nanoparticles for FRET Sensors. The FRET sensor is composed of three parts: the SAfunctionalized AgNP-1, the fluorophore (TAMRA)-carrying 4493

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Figure 1. (A) UV−vis peak of AgNP-1, AgNP-2, and AuNP; TEM images of SA-functionalized nanoparticles: (B) AgNP-1, (C) AgNP-2, and (D) AuNP.

time-consuming but with high yield. In order to decrease the time for functionalization, we further developed an approach based on streptavidin−biotin interaction for immobilizing oligonucleotides on the surface of silver nanoparticle. Protein streptavidin (SA) was bound with silver nanoparticle through electrostatic adhesion to prepare streptavidin-functionalized silver nanoparticle; then, biotin-modified aptamers were bound by streptavidin−biotin interaction, and BHQ-2 carrying strands were bound by hybridization to form the silver nanoparticlebased FRET sensor (Scheme 1). Zeta potential was measured to determine the change in the electric charge on the surface of the nanoparticles when the functionalization with protein streptavidin and oligonucleotides was accomplished. The zeta potential of SA-functionalized AgNP-1 decreased from −23.4 to −24.6 mV (n = 3) when binding with the aptamer duplex occurred. The decrease of zeta is consistent with the binding of oligonucleotides since oligonucleotides are negatively charged. In the same way, AgNP-2 and AuNP were functionalized with aptamer duplex. UV−vis spectrum was used to measure the absorption spectra of the different nanoparticles. The AgNP-1, AgNP-2, and AuNP all showed a different localized surface plasmon resonance peak. The maximum absorbance values for AgNP-1, AgNP-2, and AuNP were 465, 400, and 524 nm, respectively (Figure 1A), indicating different optical property and different enhanced fluorescence effect on nearby fluorescent dyes. In addition, the transmission electron microscope (TEM) was used to characterize the size and

aptamers, and the quencher (BHQ-2)-carrying strands. The TAMRA-carrying aptamers and BHQ-2-carrying strands form a duplex by hybridization, and this results in the FRET effect where the fluorescence of TAMRA is quenched by BHQ-2. SAfunctionalized AgNP-1 was synthesized to bind with TAMRAcarrying aptamers, which were modified with biotin on the 3′ end and fluorescent dyes TAMRA on the 5′ end, and BHQ-2carrying strands to form the silver nanoparticle-based FRET sensor. The quencher BHQ-2 would quench the fluorescence of TAMRA, thus lowering the background signal. When the PDGF-BB was added to the sensor, the BHQ-2-carrying strands were displaced and dissociated from the TAMRAcarrying aptamers resulting in the recovery of the fluorescence intensity of TAMRA-carrying aptamers. The displacement of BHQ-2-carrying strands causes a structural change to the aptamer bound with PDGF-BB. The change of conformation in the structure of aptamers shortens the distance between the fluorescent dye TAMRA and silver nanoparticles. Therefore, the silver nanoparticle could further enhance the fluorescence intensity of TAMRA by its potential fluorescence enhancement effect on the nearby fluorescent dyes.35,36,39,40 Here, the silver nanoparticle enhances the sensitivity of the FRET-based sensor. In our previous study,35,36,38−40 oligonucleotides (ssDNA, aptamer) with a sulfhydryl group at one end were used to functionalize silver nanoparticle to form a oligonucleotidefunctionalized silver nanoparticle. In this case, the self-assembly of the sulfhydryl group on the surface of silver nanoparticle was 4494

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Figure 2. (A) Excitation (a) and emission (b) spectrum of donor, the absorption spectrum (c) of acceptor. (B) The fluorescence intensity with target (F)/fluorescence intensity without target(F0) at different dilution ratio of SA-functionalized AgNP-1 for 60 min at 37 °C. (C) F/F0 at different incubation times of PDGF-BB at 37 °C. (D) F/F0 at different reaction temperatures; the concentration of PDGF-BB was 200 ng/mL.

of FRET sensor were examined since several factors are known to affect the detection efficiency of the FRET sensor. Therefore, the optimization of every parameter was important for the development of an effective and sensitive FRET sensor. For stability of proteins, BSA was always used as a stabilizer for proteins under storage and upon usage. BSA (1 mg/mL) was also used to dilute PDGF-BB since the aptamer in the sensor was highly specific toward PDGF-BB without any interference from BSA. Therefore, 1 mg/mL BSA was used to establish the fluorescence reference intensity value, F0. The fluorescence intensity ratio (F/F0 = fluorescence intensity with PDGF-BB/ fluorescence intensity with BSA) was used to describe the change caused by the concentration of PDGF-BB. A higher F/ F0 indicated a more sensitive FRET sensor. The ratio F/F0 = 1 implies that PDGF-BB is not bound with the aptamers and therefore would not denature the BHQ-2-carrying strands from the TAMRA-carrying aptamers. If F/F0 > 1, PDGF-BB displaces the BHQ-2-carrying strands, and with the increase of PDGF-BB concentration, F/F0 also increased. On the other hand, when F/F0 < 1, the nanoparticle quenches the fluorescence intensity of TAMRA. Here, the concentration of SA-functionalized AgNP-1 was optimized. According to the fabrication procedure of FRET sensor, 1 mL of SA-functionalized AgNP-1 (different dilution ratio) was mixed with 200 μL of 1 μM Apt-59bp and 200 μL of 2 μM BHQ-2 (15bp) for 60 min at 37 °C. After centrifugation, 50 μL of AgNP-1-based FRET sensor was mixed with PDGF-BB (50 μL, 200 ng/mL) in microwell plates for 1 h. Then, the plates were scanned

micrograph of SA-functionalized AgNP-1, AgNP-2, and AuNP. The diameter of SA-functionalized AgNP-1 was D = 59.1 ± 6.5 nm (N = 54), SA-functionalized AgNP-2 was D = 20.7 ± 3.3 nm (N = 69), and SA-functionalized AuNP was D = 26.6 ± 2.1 nm (N = 64). AgNP-1 nanoparticle has a larger size than that of AgNP-2 and AuNP, while the size difference of AgNP-2 and AuNP was smaller. Optimization of Silver-Enhanced FRET Sensor. The absorption and emission spectrum of the donor and the absorption spectrum of the acceptor are shown in Figure 2A. FRET response is associated with the overlap between the donor emission and acceptor absorption spectrum and the donor−acceptor distances (R) being smaller than the Förster distance (R0). The TAMRA-carrying aptamers hybridized with BHQ-2-carrying strands enables the donor (TAMRA) to be in close proximity to the quencher (BHQ-2), thus quenching the fluorescence effect. When the absorption coefficient of BHQ241 was ε580 nm= 2.35× 104 cm−1 M−1, the quantum yield of TAMRA42 was Φ = 0.076; on the basis of the equations11,22 R0 = 0.21[κ2n−4ΦJ(λ)]1/6 and E = R06/(R06 + R6), where κ is the orientation factor (κ2 = 2/3 for random collisions), n is the index of refraction (n = 1.33 in an aqueous medium), and the overlap integral J(λ) is computed on a program that overlays the normalized emission of the donor and absorption spectra of the acceptor, J(λ) = 2.43 × 1015 cm−1 M−1 nm4, the critical transfer distance R0 (in units of Å) was calculated as 38.73 Å and the actual distance R was calculated as 2.86 nm when the FRET efficiency was E = 0.86. The conditions for the formation 4495

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Figure 3. (A) F/F0 at different lengths of BHQ-2-carrying strands. (B) F/F0 at different lengths of TAMRA-carrying aptamers; the concentration of PDGF-BB was 200 ng/mL.

under BioTek instruments, and the fluorescence intensity data were collected. The excitation wavelength was λex = 532 nm, and emission wavelength was λem = 582 nm. The change in the F/F0 ratio versus concentrations is shown in Figure 2B−D. The SA functionalized AgNP-1 was mixed with different volumes of BSA in order to decrease its concentration and establish the optimal concentration for the FRET sensor. The dilution ratio was equal to (V + VBSA)/V, where V is the volume of SA functionalized AgNP-1 and VBSA is the volume of BSA. With the increase of dilution ratio (1-fold, 2-fold, 4-fold, 8-fold, 16fold), the concentration of SA functionalized AgNP-1 decreases proportionately (1-fold, 1/2-fold, 1/4-fold, 1/8-fold, 1/16fold). When the SA-functionalized AgNP-1 was diluted with 1 mg/mL BSA by 2-fold (the volume ratio of BSA to SAfunctionalized AgNP-1 was 1:1), the F/F0 ratio was almost equal to that of undiluted SA-functionalized AgNP (without BSA). The F/F0 ratio decreased with the increase in dilution ratio. Therefore, SA-functionalized AgNP-1 with 2-fold dilution ratio was chosen for the next experiments. Second, the incubation time between FRET sensor and PDGF-BB was estimated. The F/F0 ratio increased with the increase in incubation time, and the highest was observed at 70 min. Therefore, 70 min incubation time was chosen for the next experiments. Third, the temperature between FRET sensor and PDGF-BB was estimated. The F/F0 ratio increased with temperature, and the highest occurred at 47 °C. Therefore, 47 °C was chosen for the next experiments. However, 42 or 37 °C are also suitable temperatures for detection. For convenience, 42 or 37 °C could also be chosen for detection. In conclusion, the optimal conditions for the detection of PDGF-BB was 2-fold dilution of SA-functionalized AgNP-1, a 70 min incubation time, and a 47 °C incubation temperature. The hybridization efficiency of BHQ-2-carrying strands and TAMRA-carrying aptamers upon the detection ability of the FRET sensor was also investigated. The hybridization efficiency between BHQ-2-carrying strands and TAMRA-carrying aptamers was determined from the length of BHQ-2-carrying strands. The longer the BHQ-2-carrying strand, the stronger is the hybridization efficiency, resulting in more BHQ-2-carrying strands with high quenching efficiency. Therefore, the length of BHQ-2-carrying strands and TAMRA-carrying aptamers were estimated. The fluorescence intensities of the FRET sensor with different lengths of BHQ-2-carrying strands are shown in Figure 3A, and the optimal length was 12 bp. When the length of the BHQ-2-carrying strand was 9 bp, the hybridization

efficiency was weak with lower quenching efficiency, although the BHQ-2-carrying strands could easily dissociate from TAMRA-carrying aptamers by PDGF-BB. The lower quenching efficiency resulted in a higher background. The higher fluorescence intensity of BSA resulted in lower F/F0 ratio. When the length of BHQ-2 carrying strand was 15 bp, the hybridization efficiency was stronger with higher quenching efficiency resulting in lower background. However, at this length, the BHQ-2-carrying strand does not easily dissociate from TAMRA-carrying aptamers and is not easily displaced by PDGF-BB resulting in a lower F/F0 ratio. When the length of BHQ-2 carrying strand was 12 bp, the equilibrium between hybridization efficiency and dissociation efficiency was established and resulted in a higher F/F0 response. Therefore, the 12 bp length of BHQ-2-carrying strands was used for the following experiments. In addition, the length of TAMRA-carrying aptamers also affected the FRET efficiency as silver nanoparticles could enhance the fluorescence intensity of TAMRA at optimal distance. When the BHQ-2-carrying strands are dissociated from TAMRA-carrying aptamers by PDGF-BB, the distance between TAMRA and silver nanoparticle also changed, and this could be controlled by the length of TAMRA-carrying aptamers. Therefore, changing the length of the TAMRAcarrying aptamers would result in different fluorescence enhancement effects of the sensor. When the length of TAMRA-carrying aptamers was 53 bp, silver nanoparticle shows better fluorescence enhancement effect on TAMRA, as shown in Figure 3B. The distance between the SA-functionalized AgNP-1 and the TAMRA was 53 bp (approximately 0.34 nm × 53 = 18.0 nm) without PDGF-BB, while the distance became 18 bp (approximately 0.34 nm × 18 = 6.1 nm) after binding PDGF-BB. Superiority of Silver-Enhanced FRET Sensor. Different nanoparticle-based FRET sensors were fabricated and were used to compare with AgNP-1-based FRET sensor. Silver nanoparticle with size of 21 nm and gold nanoparticle with size of 27 nm were used as the support for binding with TAMRAcarrying aptamers and BHQ-2 carrying strands, and these nanoparticles produce the AgNP-2-based FRET sensor and AuNP-based FRET sensor, respectively. Preparation for these two sensors followed the same procedure as for AgNP-1-based FRET sensor. The F/F0 ratio for the four different FRET sensors at different PDGF-BB concentrations in the range of 15.6−500 ng/mL is shown in Figure 4. Results shows that the 4496

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addition, the longer distance between fluorescent dyes and gold nanoparticles, the weaker is the quenching efficiency. Even though silver nanoparticle has been widely used to enhance the fluorescence intensity of nearby fluorescent dyes, this study presents the first attempt to develop a AgNP-based FRET system for protein detection. The intensity response F/F0 to PDGF-BB by AgNP-1-based FRET sensor was also studied, and the results are shown Figure 5A. The F/F0 ratio increased with the PDGF-BB concentration in the range of 12.5−500 ng/mL, and a linear relationship between F/F0 and PDGF-BB concentration in the range of 100−500 ng/mL was observed. The specificity of the FRET sensor toward PDGF-BB was investigated in the presence of two other abundant proteins in blood (human serum albumin, immunoglobulin G). The fluorescence intensity ratios F/F0 for IgG and HSA were much lower than that of PDGF-BB as shown in Figure 5B. The presence of 1000-fold IgG and HSA (100 μg/mL) did not show any interference with the PDGFBB detection when PDGF-BB concentration was 100 ng/mL. Then, PDGF-BB in serum was tested to examine whether the complex matrix affected the silver nanoparticle-based FRET sensor. The ability of 1% serum to displace BHQ-2 carrying strand was weak compared to PDGF-BB as shown in Figure 5B, and the recovery of PDGF-BB in 100-fold diluted human serum (1% serum) was calculated as 105%, suggesting no interference from 1% serum matrix. The AgNP-based FRET sensor showed great potential for future biomarker development in a complex matrix such as serum samples. Determination of PDGF-BB by AgNP-Enhanced FRET Sensor. In order to further enhance the sensitivity of the assay, before determination of PDGF-BB at lower concentration, the volume of PDGF-BB was examined when PDGF-BB concentration was 25 ng/mL. Results are shown in Figure 6A. The F/F0 ratio of the sensor increased with PDGF-BB volume in the range of 20−170 μL, and 170 μL was chosen to investigate the region of linear relationship. At lower concentrations, increasing the volume of the target enhances the sensitivity of AgNP-enhanced FRET sensor. As volume increases, more targets tend to bind with sensor, resulting in the increase in F/F0 ratio. This lowers concentration of target that could be detected and hence lowers the detection limit. The detection ability of FRET sensor with AgNP-1 and without AgNP-1 was compared, and the results are shown in Figure 6B. The F/F0 ratio of AgNP-1-based FRET sensor increased when

Figure 4. F/F0 of FRET sensors with different nanoparticles at different concentrations of PDGF-BB, AgNP-1-based FRET sensor, AgNP-2-based FRET sensor, AuNP-based FRET sensor, and bare FRET sensor.

F/F0 ratio of the AgNP-1-based FRET sensor was highest in the concentration range of 31.2−500 ng/mL. This can be attributed to the fluorescence enhancement effect of silver nanoparticle on TAMRA. Results from silver nanoparticles with sizes of 59 and 21 nm both show enhanced F/F0 ratio. Silver nanoparticles with larger size have higher fluorescence enhancement effect on neighboring fluorescent dyes, and this was also reported in other studies.43,44 Therefore, the optimization of the size of silver nanoparticles for fluorescence enhancement is important in achieving optimum enhancement effect. At the same time, it is also known that nanoparticles with larger sizes tend to be unstable and difficult for functionalization. Therefore, stable silver nanoparticles with two sizes (59 and 21 nm) were synthesized and functionalized with aptamers. In addition, nanoparticles with sizes smaller than 5 nm tend to display very weak plasmon resonance which may quench the adjacent fluorescent dyes.45 Therefore, in our assay, the size of the nanoparticles was greater than 5 nm in order to avoid the quenching effect due to small size nanoparticles. However, the F/F0 ratio of gold nanoparticle with size of 27 nm was also found to decrease. The decreased F/F0 ratio could be attributed to the quenching effect of gold nanoparticles on TAMRA. Gold nanoparticles of small size are commonly used as a quencher in FRET systems whereby it quenches the fluorescence intensity of the nearby fluorescent dyes.14−17 The larger size of gold nanoparticles, the weaker is the quenching efficiency. In

Figure 5. (A) Response curve of F/F0 to different PDGF-BB concentrations; the inset is the linear relationship between F/F0 and concentration. (B) The specificity 100 ng/mL PDGF-BB and 100 μg/mL IgG and HSA. 4497

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Figure 6. (A) F/F0 to different volumes of PDGF-BB. (B) F/F0 of FRET sensor without and with AgNP-1; the inset is the linear relationship between F/F0 and concentration when PDGF-BB concentration is in the range of 6.2−50 ng/mL.

Research Groups (NO. 21121091). M. Vuki’s revision on the manuscript is also acknowledged.

PDGF-BB concentration was in the range of 0.8−50 ng/mL, and a linear response was observed for PDGF-BB concentration in the range of 6.2−50 ng/mL (Figure 6B, inset). The detection limit was determined as 0.8 ng/mL (32.3 pM). However, the F/F0 ratio of FRET sensor without AgNP-1 increased in the range of 12.5−50 ng/mL, and the detection limit was 12.5 ng/mL. The sensitivity increased 16-fold with AgNP-1-based FRET sensor. The assay clearly show improved sensitivity and specificity by incorporating three elements: the FRET effect between TAMRA-carrying aptamers and BHQ-2carrying strands, the strong capture ability of aptamer, and the fluorescence enhancement effect of silver nanoparticle on TAMRA.

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CONCLUSION A AgNP-based FRET sensor for protein detection was successfully developed for the detection of PDGF-BB. The newly developed AgNP-based FRET sensor showed good selectivity and sensitivity toward the target species in the presence of other proteins or in the complex matrix. Compared to FRET sensor on gold nanoparticle and bare FRET sensor, the silver nanoparticle-based FRET sensor clearly showed remarkable advantages in terms of simplicity and sensitivity. The sensitivity was increased 16-fold compared to bare FRET sensor with the detection limit of 0.8 ng/mL. The silver nanoparticle-based FRET sensor was simple, inexpensive, and convenient for protein detection. In addition, the principle of this assay could be extended to other proteins or new biomarkers for disease diagnosis and fundamental research.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel/Fax (+)00862583595835. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support of the National Basic Research Program of China (973 Program, 2011CB911003) and National Natural Foundation of China (Grant No. 21175066 and 21227009), Jiangsu Province Science and Technology Support Program (No. BE2011773) and Research Foundation of Jiangsu Province Environmental Monitoring (No. 1116), and the National Science Funds for Creative 4498

dx.doi.org/10.1021/ac400047d | Anal. Chem. 2013, 85, 4492−4499

Analytical Chemistry

Article

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