Biosensors and Bioelectronics 63 (2015) 178–184
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Nicking enzyme and graphene oxide-based dual signal amplification for ultrasensitive aptamer-based fluorescence polarization assays Yong Huang a,b,n, Xiaoqian Liu a,b, Liangliang Zhang a, Kun Hu a, Shulin Zhao a,b,n, Baizong Fang b, Zhen-Feng Chen a, Hong Liang a,b,n a Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), Guangxi Normal University, Guilin 541004, China b School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 May 2014 Received in revised form 16 July 2014 Accepted 17 July 2014 Available online 22 July 2014
In this work, two different configurations for novel amplified fluorescence polarization (FP) aptasensors based on nicking enzyme signal amplification (NESA) and graphene oxide (GO) enhancement have been developed for ultrasensitive and selective detection of biomolecules in homogeneous solution. One approach involves the aptamer-target binding induced the stable hybridization between an aptamer probe and a fluorophore-labeled DNA probe linked to GO, and forms a nicking site-containing duplex DNA region due to the enhancement of base stacking. The second analytical method involves the target induced the assembly of two aptamer subunits into an aptamer-target complex, and then hybridizes with a fluorophore-labeled DNA probe linked to GO, forming a nicking site-containing duplex DNA region. The formation of the duplex DNA region in both methods triggers the NESA process, resulting in the release of many short DNA fragments carrying the fluorophore from GO, generating a significant decrease of the FP value that provides the readout signal for the amplified sensing process. By using the NESA coupled GO enhancement path, the sensitivity of the developed aptasensors can be significantly improved by four orders of magnitude over traditional aptamer-based homogeneous assays. Moreover, these aptasensors also exhibit high specificity for target molecules, which are capable of detecting target molecule in biological samples. Considering these qualities, the proposed FP aptasensors based NESA and GO enhancement can be expected to provide an ultrasensitive platform for amplified analysis of target molecules. & 2014 Elsevier B.V. All rights reserved.
Keywords: Nicking enzyme Graphene oxide Fluorescence polarization Aptasensors Amplified analysis
1. Introduction The development of aptasensors has attracted substantial research efforts, and a variety of techniques have been exploited to develop aptasensors (Citartan et al., 2012; Liu et al. 2009; Tan et al., 2013) for detection of various targets. Among these methods, fluorescence polarization (FP) analysis offers an appealing approach for the detection of aptamer substrates due to its simplicity and rapidness (Deng et al., 2006, 2007; Zhang et al., 2011a). Many FP aptasensors have been developed for the detection of proteins (Zhang et al., 2011b, 2012a; Zou et al., 2012), small molecules (Perrier et al., 2010; Ruta et al., 2009; Zhang et al., 2012b; Zhao et al., 2014), and cancer cells (Deng et al., 2010). However, as with n Corresponding authors at: Guangxi Normal University, School of Chemistry and Pharmacy, Yucai Road 15, Guilin 541004, China. Tel.: þ 86 773 5845973; fax: þ 86 773 5832294. E-mail addresses: [email protected] (Y. Huang), [email protected] (S. Zhao), [email protected] (H. Liang).
http://dx.doi.org/10.1016/j.bios.2014.07.036 0956-5663/& 2014 Elsevier B.V. All rights reserved.
other signaling transduction approaches, the development of FP aptasensors is often accompanied by unsatisfactory sensitivities of the systems that are controlled by relatively low association constants of aptamers to their substrates. Several amplification strategies to improve the sensitivities of FP aptasensors have been developed. These include the use of gold nanoparticles (Ye and Yin, 2008), silica nanoparticles (Huang et al., 2012), graphene oxide (Liu et al., 2013a, 2013b; Yu et al., 2013), proteins (Cui et al., 2012; Zhu et al., 2012), and DNA-protein hybrid nanowires (Yang et al., 2013) as FP enhancers. Although enhanced sensitivities were demonstrated by these amplification strategies, greater sensitivity and specificity are frequently required, particularly when working with limited amounts of sample material or when target level is extremely low. Nicking enzymes (NEs) are a special group of restriction endonucleases that can cleave one strand of a duplex DNA. This function has been used to develop different amplified NEs-based detection platforms that involve cyclic NEs-catalyzed cleavage of DNA by target recognition. For example, the cyclic NEs-catalyzed
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Tb aptamer subunit-1 (ATA-1):5′-GGGACCTCAGCGCAGTCCGT GGTAGGGC-3′ Tb aptamer subunit-2 (ATA-2): 5′-TAGGTTGGGGTGACTGC-3′ DNA-1: 5′-FAM-GC↓TGAGGATTTTTTTTTTTT-3′ DNA-2: 5′-FAM-GC↓TGAGGTTTTTTTTTTTTT-3′ DNA-3: 5′-FAM-GC↓TGAGGTCTTTTTTTTTTT-3′ DNA-4: 5′-FAM-GC↓TGAGGTCCTTTTTTTTTT-3′
cleavage of specific DNA by nucleic acids was used to amplify nucleic acid detection (Bi et al., 2010; Kong et al., 2011; Liu et al., 2011; Lin et al., 2011; Zou et al., 2011; Yin et al., 2013). Also, the NEs-catalyzed recycling of aptamer-substrate complexes was used for the amplified fluorescence and colorimetric sensing of biomolecules (Huang et al., 2013; Hun et al., 2013; Li et al., 2012; Xue et al., 2012; Zheng et al., 2012). In addition, NEs were also used as biocatalytic amplifiers for monitoring enzyme activities (Chen et al., 2013; Liu et al., 2014). However, to the best of our knowledge, such NEs-assisted signal amplification systems have not been investigated in any attempts for their use in a FP biosensor. Graphene oxide (GO) has found versatile applications in biological studies due to its unique optoelectronic properties and excellent biocompatibility (Chen et al., 2010a, 2010b; Loh et al., 2010). Specifically, GO has been regarded as a kind of superquencher that can effectively quench the fluorescence of a range of dyes (Chang et al. 2010; Lu et al., 2010a, 2010b). Based on this superquenching effect, GO has been employed to develop nanosensors for sensitive detection of nucleic acids (Dong et al., 2010; He et al., 2010; Liu et al., 2013a, 2013b; Lu et al., 2009; Zhang et al., 2013), aptamer substrates (Hu et al., 2013; Li et al., 2013; Lu et al., 2010a, 2010b; Wang et al., 2013; Zhang et al., 2011c), and enzyme activities (Li et al., 2011; Zhang et al., 2011d; Zhu et al., 2013), etc. Besides this property, recent studies suggest that GO can be used as an effective FP enhancer due to its extraordinarily larger volume. For example, Yang's group first reported the use of GO as an enhancer for the development of FP aptasensors for sensitive detection of ATP (Liu et al., 2013a, 2013b). Similarly, a GOenhanced FP strategy was used for DNAzyme-based assay of metal ions by Huang and co-workers (Yu et al., 2013). However, exploration of GO with FP analysis still remains at a very early stage. In this work, we report a new strategy for the development of amplified FP aptasensors for ultrahighly sensitive and selective detection of target molecules in homogeneous solution based on nicking enzyme signal amplification (NESA) and GO enhancement. Compared with traditional homogeneous aptasensors, the detection sensitivity of the developed aptasensors can be significantly improved by four orders of magnitude by using the NESA coupled GO enhancement approach. Moreover, the proposed aptamerbased sensing assays are conducted in aqueous solution, and not requiring separation and other troublesome procedures, which is very simple and convenient. With the use of adenosine (A) and thrombin (Tb) as model analytes, these new sensing platforms exhibit very high detection sensitivity, high specificity and wide dynamic ranges over six orders of magnitude. Furthermore, the suitability of the proposed method for biological sample analysis has also been demonstrated.
In a typical adenosine (A) assay, the AAA probe (20 nM) and the DNA-2 probe (85 nM) were incubated with GO (100 μg/mL) in 240 μL Tris–HCl buffer (20 mM Tris–HCl, 50 mM NaCl, 10 mM Mg2 þ , and 50 mM K þ , pH 7.9) for 1 min at room temperature. After that, 10 μL the above Tris–HCl buffer containing Nb.BbvCI (0.5 U/μL) and different concentrations of A was added, and incubated the mixture at 37 °C for another 1 h. The obtained sample solution was used for FP measurements. Control experiments were performed under otherwise identical conditions but in the absence of GO or Nb.BbvCI, or in the absence of both GO and Nb.BbvCI. All experiments were repeated three times. FP of the sample solutions was measured by using the L-format configuration and FluorEssence™ software with constant wavelength analysis to achieve a FP value. The G factor was initially set to zero, to let the system measure G automatically. The FP value was also calculated automatically by the instrument. The integration time was set to 3 s for the FP measurements. Over five FP measurements were taken each time, and they were then averaged for further data processing.
2. Experimental
2.4. Thrombin assay with FP aptasensors
2.1. Materials and reagents
In a typical thrombin (Tb) assay, the ATA-1 probe (20 nM), the ATA-2 probe (20 nM), and the DNA-2 probe (85 nM) were incubated with GO (100 μg/mL) in 240 μL Tris–HCl buffer (20 mM Tris– HCl, 50 mM NaCl, 10 mM Mg2 þ , and 50 mM K þ , pH 7.9) for 1 min at room temperature. Afterward, 10 μL the Tris–HCl buffer (20 mM Tris–HCl, 50 mM NaCl, 10 mM Mg2 þ , and 50 mM K þ , pH 7.9) containing Nb.BbvCI (0.5 U/μL) and different concentrations of Tb was added, and incubated the mixture at 37 °C for another 1 h. The resulting sample solution was used for FP measurements. Control experiments were performed under otherwise identical conditions but in the absence of GO or Nb.BbvCI, or in the absence of both GO and Nb.BbvCI. All experiments were repeated three times. The procedure for FP measurements was the same as that of A detection described above.
Adenine (A), thymine (T), guanine (G), cytosine (C), thrombin (Tb), human serum albumin (HSA), human immunoglobulin G (IgG), human immunoglobulin E (IgE), thrombin (Tb), and graphene oxide (GO) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Nicking endonuclease Nb.BbvCI and human factor Xa were obtained from New England Biolabs (NEB, U.K.). All oligonucleotides were purchased from the Sangon Biotech Co. (Shanghai, China) and purified by HPLC. The sequences of the involved oligonucleotides were as follows: A aptamer (AAA): 5′-GGGACCTCAGCACCTGGGGGAGTTGCGGAGGAAGGT-3′
The italic bold sequences of the above oligonucleotides probes were the aptamer nucleotides, and the underlined sequences of AAA and ATA-1 were partly complementary to DNA-1, DNA-2, DNA-3, and DNA-4, respectively. The arrow indicated the nicking position of Nb.BbvCI. Other chemicals were of analytical grade. Water was purified by using a Milli-Q plus 185 equip from Millipore (Bedford, MA). 2.2. Apparatus Fluorescence polarization (FP) measurements were carried out using an FL3-P-TCSPC system (Jobin Yvon, Inc., Edison, NJ, USA) with 300 μL cuvette. The FP of the sample solution was monitored by exciting the sample at 490 nm and measuring the emission at 520 nm. And slits for both the excitation and the emission were set at 5 nm. 2.3. Adenosine assay with FP aptasensors
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2.5. Procedure for detection of thrombin in human plasma samples Five normal human plasma sample collected in the citrate anticoagulated tubes was kindly provided by the No. 5 People′s Hospital (Guilin, China). Fibrinogen was removed through precipitation according to the method reported in the literature (Chen et al., 2010a, 2010b). Briefly, 0.1 mL of human plasma was quickly mixed with 1.25 mL of ammonium sulfate (2 M) and 1 mL of NaCl (0.1 M). After 4 min incubation, the mixture was centrifuged and the upper supernatant solution was collected. Then, CaCl2 (0.03 M) with 8 nM human factor Xa was then added to the plasma to promote the transformation from prothrombin to thrombin. Finally, the resulting plasma diluted 100-fold with Tris–HCl buffer (20 mM Tris–HCl, 50 mM NaCl, 10 mM Mg2 þ , and 50 mM K þ , pH 7.9), and immediately used for Tb detection by the proposed FP aptasensor. The assay procedures were the same as that of the standard Tb detection noted above.
3. Results and discussion 3.1. Principle of the amplified aptamer-based FP assay for adenosine detection By taking the advantages of NESA and GO enhancement, we present a new strategy for development of amplified FP aptasensors for detection of biomolecules. Scheme 1 depicts one amplified FP aptasensor configuration that is exemplified with detection of A. This system consists of an anti-A aptamer probe (AAA), an assistant DNA probe (FAM-labeled DNA-2), GO, and nicking endonuclease Nb.Bbvc I. The AAA probe is designed with a 10-nt extension sequence at its 5′-end. The FAM-labeled DNA-2 probe is designed to be partly complementary to the short extension sequence of the AAA probe, and also contains the cleavage site of Nb.Bbvc I. In the absence of A, the AAA probe is unable to bind with the FAM-labeled DNA-2 probe because the complementary oligonucleotides are too short to promote efficient hybridization. In this case, both AAA and FAM-labeled DNA-2 probes are adsorbed onto the GO surface, thus the FAM dye exhibits very high FP value (P) due to the extraordinarily larger volume of GO. When A is introduced to the system, the AAA probe binds the A target to form an A/aptamer complex with a duplex DNA region, and thus the enhancement of base stacking (Cai et al., 2013). This allows the A/aptamer complex to hybridize with a FAM-labeled DNA-2 probe associated with GO, resulting in another DNA duplex region containing the recognition sequence of Nb.Bbvc I. This triggers the selective enzymatic cleavage of the FAM-labeled DNA-2 probe by Nb.Bbvc I, resulting in the release of a short DNA fragment carrying the FAM dye from the GO surface and the decrease of the P value. In addition, the Nb.Bbvc I-catalyzed cleavage of the FAM-labeled DNA-2 probe releases the intact A/ aptamer complex that can then hybridize with another FAMlabeled DNA-2 probe associated with GO to initiate the cleavage of the FAM-labeled DNA-2 probe. Thus, Nb.Bbvc I recycles the
Fig. 1. FP values in the absence (black) and presence (red) of 400 nM adenosine with different sensing systems. (A) The AAA(20 nM)/DNA-2(85 nM)/GO (100 μg/mL)/Nb.BbvCI(0.5 U/μL) system; (B) the AAA(20 nM)/DNA-2(85 nM)/Nb. BbvCI (0.5 U/μL) system; (C) the AAA(20 nM)/DNA-2(85 nM) system; (D) the AAA (20 nM)/DNA-2(85 nM)/GO(100 μg/mL) system. Error bars were derived from N¼ 3 experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
A/aptamer complex, leading to the continuous cleavage of the FAM-labeled DNA-2 probes linked to GO and the release of many short DNA fragments carrying the FAM dye from GO, generating a substantial decrease of the P value, and thus achieving the target assisted dual signal amplification. Of note, the introduction of GO and Nb.Bbvc I provides significant signal amplification, which can substantially improve the detection sensitivity of the FP aptasensor compared with traditional homogeneous aptasensors. 3.2. Feasibility study To verify the feasibility of our design, the FP assay for detection of 400 nM A at different conditions was investigated, and the results obtained are shown in Fig. 1. With the AAA/DNA-2 system, the P value increased slightly upon the addition of A. However, control experiments revealed that no changes of the P value occurred when using DNA-2 only for A detection. This suggested the formation of stable A-mediated complex between the AAA probe and the FAM-labeled DNA-2 probe. When Nb.Bbvc I was introduced to the AAA/DNA-2 system, the P value decreased significantly upon the addition of A, indicating that target assisted Nb.Bbvc I-catalyzed cleavage of the FAM-labeled DNA-2 probe had taken place and generated short DNA fragments carrying the FAM dye. When the AAA/DNA-2/Nb.Bbvc I/GO system was used for A detection, the background P value was greatly enhanced to 416, which was about 4-fold higher than the situation without GO. Moreover, the ΔP value of this system for A detection is approximately 5 times higher than that of the system without GO enhancement and 36 times higher that of the system without
Scheme 1. Amplified FP analysis of adenosine using GO enhancement and nicking enzyme-aided recycling of the adenosine–aptamer complex.
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both NESA and GO enhancement. However, control experiments revealed that no apparent change of the P value occurred when the AAA/DNA-2/GO system was used for A detection. The significant enhancement of the ΔP value was attributed to the fact that GO could greatly enhance the background P value and short fluorescent DNA fragments with faster rotation were released from GO by target assisted Nb.Bbvc I-catalyzed cyclic cleavage of the FAM-labeled DNA-2 probe linked to GO. These results demonstrated that the introduction of GO and Nb.Bbvc I could significantly amplify the reporting signal for the FP aptasensor.
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GO. Thus, the background FP value would be low. On the other hand, too much of GO would inhibit the release of the cleaved products from GO and result in a poor reporting signal. Thus, it is necessary to optimize the amount of GO to give the largest ΔP enhancement. Fig. S3 shows the effects of different concentrations of GO on the ΔP value. As the concentration of GO increased, the ΔP value increased gradually upon the addition of A. The largest ΔP value was obtained when 100 μg/mL GO was employed. Further increasing GO concentration, the ΔP value decreased, which might be attributed to the inhibition of the release of the cleaved products from GO by high concentration of GO.
3.3. Optimization of assay conditions 3.4. Adenosine assay For such FP amplifying design, the complementary sequence of the AAA probe to the assistant DNA probe is a critical factor. On the one hand, the FP signal amplification promoted only in the presence of A as we designed, so if the complementary bases is too long, it may initiate the FP signal amplification both in the presence and absence of A. On the other hand, too short complementary bases would not initiate the FP signal amplification even in presence of A. Therefore, it is essential to optimize the complementary bases between the AAA probe and the assistant DNA probe. Our AAA probe was designed with a 10-nt extension sequence at its 5′-end, four assistant DNA probes that contains different complementary bases to the AAA probe were tested. Fig. S2 shows the effects of four assistant DNA probes with 7–10 complementary bases of the AAA probe on the P value of the amplified sensing system in the absence and presence of A. As can be seen, the DNA-2 probe with 8-nt complementary bases provided the best signal to background ratio. This was attributed to the fact that the DNA-2 probe with 8-nt complementary bases could stabilize the hybridization between the AAA probe and the assistant DNA probe and then triggered an effective NESA process only in the presence of A. Therefore, the DNA-2 probe was selected as the optimal assistant DNA probe for the following assays. In addition, the amount of GO also significantly affected the reporting FP signal. On one hand, if the amount of GO is too small, the FAMlabeled DNA probe will not be adsorbed enough on the surface of
The proposed amplified FP aptasensor for A detection is highly sensitive. Fig. 2, curve a, shows the FP responses of the proposed amplified sensing system upon analyzing different concentrations of A, for a fixed reaction time-interval of 1 h. With increasing of the A concentration, the ΔP value increased gradually, consistent with the enhanced cleavage of the FAM-labeled DNA-2 probe linked to GO by Nb.Bbvc I-recycled the A/aptamer complex and the release of more amounts of short DNA fragments carrying the FAM dye from GO. Interestingly, as shown in Fig. 2, inset, the plots of the ΔP value versus A concentration in the range of 4 pM–10 μM showed good linear relationships (R2 ¼0.9981). This amplified FP aptasensor allowed the detection of A with a detection limit corresponding to 2.0 pM, which was at least four orders of magnitude lower than that of traditional homogeneous aptasensors, and two orders of magnitude lower than that of the reported amplified aptamer-based homogeneous assays (Table S1). In contrast, when the FP aptasensor without GO enhancement was used, a sensitivity that corresponds to an A concentration of 0.2 nM was obtained (Fig. 2, curve b); when the FP aptasensor without NESA and GO enhancement was used, a sensitivity that corresponds to an A concentration of 200 nM was obtained (Fig. 2, curve c). The substantial sensitivity improvement of this amplified FP aptasensor was attributed to the reasons as follows: 1) GO with large volume could greatly enhance the background P value when the
Fig. 2. Plots of FP changes as a function of adenosine concentrations using different sensing systems. (a) The AAA(20 nM)/DNA-2(85 nM)/GO(100 μg/mL)/ Nb.BbvCI(0.5 U/μL) system; (b) the AAA(20 nM)/DNA-2(85 nM)/Nb.BbvCI (0.5 U/μL) system; (c) the AAA(20 nM)/DNA-2(85 nM) system. Inset. The derived calibration curve corresponding to the △P value of the AAA/DNA-2/ GO/Nb.BbvCI system. Error bars were derived from N ¼3 experiments.
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FAM-labeled DNA-1 probe was adsorbed onto GO; 2) the A target assisted Nb.Bbvc I-catalyzed cleavage of the FAM-labeled DNA-1 probe resulted in the release of many short DNA fragments carrying the FAM dye with faster rotation. This amplified FP aptasensor for A detection was also specific. To evaluate this property, we tested the response of the assay to the target A and several analogue molecules: cytidine (C), guanosine (G), and thymine (T). It was found that the specific target A led to an increase in the ΔP value, and no obvious change of the ΔP value was observed in the assay for all analogue molecules as compared to the background (Fig. S4). These results clearly demonstrated the high specificity of our amplified FP aptasensor. In addition, the amplified FP aptasensor also exhibited acceptable precision, and the variation coefficients (CVs) for five repetitive measurements of 40 pM, 8 nM, and 0.8 μM A were 1.8–5.4%, which were comparable to the reported methods (Cai et al., 2013; Chen et al., 2008). 3.5. Thrombin detection To illustrate the generality of our strategy, we applied this strategy to develop another amplified FP aptasensor for Tb detection (Scheme 2). Thrombin (Tb) was used as a model analyte. This sensing platform involves the use of the aptamer subunits approach. The two anti-Tb aptamer subunits ATA-1 and ATA-2 are used, and ATA-1 includes a 10-nt extension sequence at its 5′end that is designed to be partly complementary to the FAMlabeled DNA-2 probe. The aptamer subunits and the FAM-labeled DNA-2 probe can coexist stably in the absence of Tb, and they are adsorbed onto GO, thus the FAM dye exhibits very high P value. However, the addition of Tb results in the assembly of two aptamer subunits into a Tb/aptamer complex with a duplex region, which promotes the hybridization between the FAM-labeled DNA2 probe and the Tb/aptamer complex to form a duplex domain containing the cleavage site of Nb.Bbvc I. This triggers the Nb.Bbvc I-catalyzed selective cleavage of the FAM-labeled DNA-2 probe, resulting in the removal of the FAM dye from GO, the release of the Tb/aptamer complex and the decrease of the P value. The released Tb/aptamer complex can then hybridize with another FAMlabeled DNA-2 probe linked to GO, and the cycle starts anew, which leads to significant amplification of the reporting signal. By monitoring the decrease in the P value, we could detect Tb with very high sensitivity. Firstly, the key factors affecting the amplified FP assay for Tb detection were optimized (Figs. S5 and S6). After that, the ability of the amplified system for Tb detection was demonstrated. As shown in Fig. 3, curve a, the amplified FP aptasensor based on NESA and GO enhancement enabled the detection of Tb with a detection limit of 1 fM, which was three orders of magnitude lower than that of the FP aptasensor without GO enhancement (0.1 pM) (Fig. 3, curve b), and five orders of magnitude lower than that of the FP aptasensor without NESA and GO enhancement (1 nM) (Fig. 3, curve c). Meanwhile, the detection limit for Tb in this work was also four orders of magnitude than that of
traditional homogeneous aptasensors and two orders of magnitude lower than that of the reported amplified aptamer-based homogeneous assays (Table S1). The sensitivity improvement of this amplified FP aptasensor for Tb detection was attributed to the fact that GO associated with the FAM-labeled DNA-2 probe could greatly enhanced the background P value, and many short DNA fragments carrying the FAM dye with faster rotation were released from GO by Tb assisted Nb.Bbvc I-catalyzed DNA cleavage reaction. The results also showed good linear relationships (R2 ¼0.9966) between the ΔP value and Tb concentration in the range of 2 fM– 200 nM (Fig. 3, inset). This amplified FP aptasensor also showed high specificity toward Tb over other non-specific proteins which were co-existing in biological samples, such as IgG, IgE, HSA and AFP (Fig. S7). Moreover, this amplified FP aptasensor also exhibited acceptable precision, and the CVs for five repetitive measurements of 20 fM, 2 pM, and 2 nM Tb were 2.2–5.6%, which were comparable to the reported methods (Yuan et al., 2011; Huang et al., 2013). 3.6. Detection of thrombin in human plasma To evaluate the application of the amplified FP aptasensor to real sample, the determination of Tb in five human plasma samples was conducted by the present method and compared with those obtained by the traditional ELISA method. As shown in Table 1, the assay results of Tb in human plasma by the amplified FP aptasensor were in an acceptable agreement with those of the ELISA method, and were also consistent with the reported levels (Chen et al., 2010a, 2010b; Wang et al., 2011). Moreover, the method also revealed the good recovery rates of standard addition from 94.2% to 105.2%. In addition, the assay precision was also evaluated by repeatedly analyzing each human plasma sample five times within a working day, and the relative standard deviations for assays were in the range of 3.4% to 4.8%. These results indicated that the proposed amplified FP aptasensor could be acceptable for quantitative assays performed in biological samples.
4. Conclusions In summary, we have developed two different configurations for developing new amplified FP aptasensors based on both NESA and GO enhancement. By the appropriate design of the aptamer probe or aptamer subunits, and a fluorophore- labeled assistant DNA probe, these configurations can be effectively applied for highly sensitive and selective detection of A or Tb (as model analytes). Furthermore, the suitability of the amplified FP aptasensor for complex biological sample analysis has also been demonstrated. These assays are quite simple and convenient, needing only mixing the GO-functionalized oligonucleotide probes, the nicking endonuclease, and the target molecule in homogeneous solution, and not requiring separation and troublesome procedures. Moreover, the sensitivity of these amplified FP
Scheme 2. Amplified FP sensing of thrombin using two aptamer subunits and nicking enzyme-catalyzed regeneration of the thrombin-aptamer complex.
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Fig. 3. Plots of FP changes as a function of thrombin concentrations using different sensing systems. (a) The ATA-1(20 nM)/ATA-2(20 nM)/DNA-2(85 nM)/GO(100 μg/mL)/Nb. BbvCI (0.5 U/μL) system; (b) the ATA-1(20 nM)/ATA-2(20 nM)/DNA-2(85 nM)/Nb.BbvCI (0.5 U/μL) system; (c) the ATA-1(20 nM)/ATA-2 (20 nM)/DNA-2(85 nM) system. Inset. The derived calibration curve corresponding to the ΔP value of the ATA-1/ATA-2/DNA-2/GO/Nb.BbvCI system. Error bars were derived from N ¼ 3 experiments. Table 1 Determination of thrombin in five human plasma samples. Sample
Present method (nM)
ELISA (nM)
Relative deviation (%)
1 2 3 4 5
86.3 102.4 119.2 103.6 96.3
89.6 97.5 110.8 108.7 103.3
3.7 5.0 7.6 4.7 6.8
aptasensors is at least four orders of magnitude higher than that of traditional unamplified aptamer-based homogeneous assays. Additionally, these approaches may also be extended to other target molecules detection by simply switching the corresponding aptamer probes or aptamer subunits. Finally, these assays can be easily carried out in 96- or 384-well plates, rendering it suitable for routine high-throughput applications. Thus, the proposed amplified FP aptasensors can be expected to provide sensitive and general platforms for amplified analysis of target molecules.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 21305021 and 21175030), the National Basic Research Program of China (No. 2012CB723501), and the Natural Science Foundations of Guangxi Province (Nos. 2012GXNSFDA385001, 2013GXNSFBA019038, and 2013GXNS FBA019044) as well as BAGUI Scholar Program and the Project of Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Education of China (CMEMR2012-A19 and CMEMR2013-C06).
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.036.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Nicking enzyme and graphene oxide-based dual signal amplification for ultrasensitive aptamer-based fluorescence polarization assays Yong Huang a,b,n, Xiaoqian Liu a,b, Liangliang Zhang a, Kun Hu a, Shulin Zhao a,b,n, Baizong Fang b, Zhen-Feng Chen a, Hong Liang a,b,n a Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), Guangxi Normal University, Guilin 541004, China b School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 May 2014 Received in revised form 16 July 2014 Accepted 17 July 2014 Available online 22 July 2014
In this work, two different configurations for novel amplified fluorescence polarization (FP) aptasensors based on nicking enzyme signal amplification (NESA) and graphene oxide (GO) enhancement have been developed for ultrasensitive and selective detection of biomolecules in homogeneous solution. One approach involves the aptamer-target binding induced the stable hybridization between an aptamer probe and a fluorophore-labeled DNA probe linked to GO, and forms a nicking site-containing duplex DNA region due to the enhancement of base stacking. The second analytical method involves the target induced the assembly of two aptamer subunits into an aptamer-target complex, and then hybridizes with a fluorophore-labeled DNA probe linked to GO, forming a nicking site-containing duplex DNA region. The formation of the duplex DNA region in both methods triggers the NESA process, resulting in the release of many short DNA fragments carrying the fluorophore from GO, generating a significant decrease of the FP value that provides the readout signal for the amplified sensing process. By using the NESA coupled GO enhancement path, the sensitivity of the developed aptasensors can be significantly improved by four orders of magnitude over traditional aptamer-based homogeneous assays. Moreover, these aptasensors also exhibit high specificity for target molecules, which are capable of detecting target molecule in biological samples. Considering these qualities, the proposed FP aptasensors based NESA and GO enhancement can be expected to provide an ultrasensitive platform for amplified analysis of target molecules. & 2014 Elsevier B.V. All rights reserved.
Keywords: Nicking enzyme Graphene oxide Fluorescence polarization Aptasensors Amplified analysis
1. Introduction The development of aptasensors has attracted substantial research efforts, and a variety of techniques have been exploited to develop aptasensors (Citartan et al., 2012; Liu et al. 2009; Tan et al., 2013) for detection of various targets. Among these methods, fluorescence polarization (FP) analysis offers an appealing approach for the detection of aptamer substrates due to its simplicity and rapidness (Deng et al., 2006, 2007; Zhang et al., 2011a). Many FP aptasensors have been developed for the detection of proteins (Zhang et al., 2011b, 2012a; Zou et al., 2012), small molecules (Perrier et al., 2010; Ruta et al., 2009; Zhang et al., 2012b; Zhao et al., 2014), and cancer cells (Deng et al., 2010). However, as with n Corresponding authors at: Guangxi Normal University, School of Chemistry and Pharmacy, Yucai Road 15, Guilin 541004, China. Tel.: þ 86 773 5845973; fax: þ 86 773 5832294. E-mail addresses: [email protected] (Y. Huang), [email protected] (S. Zhao), [email protected] (H. Liang).
http://dx.doi.org/10.1016/j.bios.2014.07.036 0956-5663/& 2014 Elsevier B.V. All rights reserved.
other signaling transduction approaches, the development of FP aptasensors is often accompanied by unsatisfactory sensitivities of the systems that are controlled by relatively low association constants of aptamers to their substrates. Several amplification strategies to improve the sensitivities of FP aptasensors have been developed. These include the use of gold nanoparticles (Ye and Yin, 2008), silica nanoparticles (Huang et al., 2012), graphene oxide (Liu et al., 2013a, 2013b; Yu et al., 2013), proteins (Cui et al., 2012; Zhu et al., 2012), and DNA-protein hybrid nanowires (Yang et al., 2013) as FP enhancers. Although enhanced sensitivities were demonstrated by these amplification strategies, greater sensitivity and specificity are frequently required, particularly when working with limited amounts of sample material or when target level is extremely low. Nicking enzymes (NEs) are a special group of restriction endonucleases that can cleave one strand of a duplex DNA. This function has been used to develop different amplified NEs-based detection platforms that involve cyclic NEs-catalyzed cleavage of DNA by target recognition. For example, the cyclic NEs-catalyzed
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Tb aptamer subunit-1 (ATA-1):5′-GGGACCTCAGCGCAGTCCGT GGTAGGGC-3′ Tb aptamer subunit-2 (ATA-2): 5′-TAGGTTGGGGTGACTGC-3′ DNA-1: 5′-FAM-GC↓TGAGGATTTTTTTTTTTT-3′ DNA-2: 5′-FAM-GC↓TGAGGTTTTTTTTTTTTT-3′ DNA-3: 5′-FAM-GC↓TGAGGTCTTTTTTTTTTT-3′ DNA-4: 5′-FAM-GC↓TGAGGTCCTTTTTTTTTT-3′
cleavage of specific DNA by nucleic acids was used to amplify nucleic acid detection (Bi et al., 2010; Kong et al., 2011; Liu et al., 2011; Lin et al., 2011; Zou et al., 2011; Yin et al., 2013). Also, the NEs-catalyzed recycling of aptamer-substrate complexes was used for the amplified fluorescence and colorimetric sensing of biomolecules (Huang et al., 2013; Hun et al., 2013; Li et al., 2012; Xue et al., 2012; Zheng et al., 2012). In addition, NEs were also used as biocatalytic amplifiers for monitoring enzyme activities (Chen et al., 2013; Liu et al., 2014). However, to the best of our knowledge, such NEs-assisted signal amplification systems have not been investigated in any attempts for their use in a FP biosensor. Graphene oxide (GO) has found versatile applications in biological studies due to its unique optoelectronic properties and excellent biocompatibility (Chen et al., 2010a, 2010b; Loh et al., 2010). Specifically, GO has been regarded as a kind of superquencher that can effectively quench the fluorescence of a range of dyes (Chang et al. 2010; Lu et al., 2010a, 2010b). Based on this superquenching effect, GO has been employed to develop nanosensors for sensitive detection of nucleic acids (Dong et al., 2010; He et al., 2010; Liu et al., 2013a, 2013b; Lu et al., 2009; Zhang et al., 2013), aptamer substrates (Hu et al., 2013; Li et al., 2013; Lu et al., 2010a, 2010b; Wang et al., 2013; Zhang et al., 2011c), and enzyme activities (Li et al., 2011; Zhang et al., 2011d; Zhu et al., 2013), etc. Besides this property, recent studies suggest that GO can be used as an effective FP enhancer due to its extraordinarily larger volume. For example, Yang's group first reported the use of GO as an enhancer for the development of FP aptasensors for sensitive detection of ATP (Liu et al., 2013a, 2013b). Similarly, a GOenhanced FP strategy was used for DNAzyme-based assay of metal ions by Huang and co-workers (Yu et al., 2013). However, exploration of GO with FP analysis still remains at a very early stage. In this work, we report a new strategy for the development of amplified FP aptasensors for ultrahighly sensitive and selective detection of target molecules in homogeneous solution based on nicking enzyme signal amplification (NESA) and GO enhancement. Compared with traditional homogeneous aptasensors, the detection sensitivity of the developed aptasensors can be significantly improved by four orders of magnitude by using the NESA coupled GO enhancement approach. Moreover, the proposed aptamerbased sensing assays are conducted in aqueous solution, and not requiring separation and other troublesome procedures, which is very simple and convenient. With the use of adenosine (A) and thrombin (Tb) as model analytes, these new sensing platforms exhibit very high detection sensitivity, high specificity and wide dynamic ranges over six orders of magnitude. Furthermore, the suitability of the proposed method for biological sample analysis has also been demonstrated.
In a typical adenosine (A) assay, the AAA probe (20 nM) and the DNA-2 probe (85 nM) were incubated with GO (100 μg/mL) in 240 μL Tris–HCl buffer (20 mM Tris–HCl, 50 mM NaCl, 10 mM Mg2 þ , and 50 mM K þ , pH 7.9) for 1 min at room temperature. After that, 10 μL the above Tris–HCl buffer containing Nb.BbvCI (0.5 U/μL) and different concentrations of A was added, and incubated the mixture at 37 °C for another 1 h. The obtained sample solution was used for FP measurements. Control experiments were performed under otherwise identical conditions but in the absence of GO or Nb.BbvCI, or in the absence of both GO and Nb.BbvCI. All experiments were repeated three times. FP of the sample solutions was measured by using the L-format configuration and FluorEssence™ software with constant wavelength analysis to achieve a FP value. The G factor was initially set to zero, to let the system measure G automatically. The FP value was also calculated automatically by the instrument. The integration time was set to 3 s for the FP measurements. Over five FP measurements were taken each time, and they were then averaged for further data processing.
2. Experimental
2.4. Thrombin assay with FP aptasensors
2.1. Materials and reagents
In a typical thrombin (Tb) assay, the ATA-1 probe (20 nM), the ATA-2 probe (20 nM), and the DNA-2 probe (85 nM) were incubated with GO (100 μg/mL) in 240 μL Tris–HCl buffer (20 mM Tris– HCl, 50 mM NaCl, 10 mM Mg2 þ , and 50 mM K þ , pH 7.9) for 1 min at room temperature. Afterward, 10 μL the Tris–HCl buffer (20 mM Tris–HCl, 50 mM NaCl, 10 mM Mg2 þ , and 50 mM K þ , pH 7.9) containing Nb.BbvCI (0.5 U/μL) and different concentrations of Tb was added, and incubated the mixture at 37 °C for another 1 h. The resulting sample solution was used for FP measurements. Control experiments were performed under otherwise identical conditions but in the absence of GO or Nb.BbvCI, or in the absence of both GO and Nb.BbvCI. All experiments were repeated three times. The procedure for FP measurements was the same as that of A detection described above.
Adenine (A), thymine (T), guanine (G), cytosine (C), thrombin (Tb), human serum albumin (HSA), human immunoglobulin G (IgG), human immunoglobulin E (IgE), thrombin (Tb), and graphene oxide (GO) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Nicking endonuclease Nb.BbvCI and human factor Xa were obtained from New England Biolabs (NEB, U.K.). All oligonucleotides were purchased from the Sangon Biotech Co. (Shanghai, China) and purified by HPLC. The sequences of the involved oligonucleotides were as follows: A aptamer (AAA): 5′-GGGACCTCAGCACCTGGGGGAGTTGCGGAGGAAGGT-3′
The italic bold sequences of the above oligonucleotides probes were the aptamer nucleotides, and the underlined sequences of AAA and ATA-1 were partly complementary to DNA-1, DNA-2, DNA-3, and DNA-4, respectively. The arrow indicated the nicking position of Nb.BbvCI. Other chemicals were of analytical grade. Water was purified by using a Milli-Q plus 185 equip from Millipore (Bedford, MA). 2.2. Apparatus Fluorescence polarization (FP) measurements were carried out using an FL3-P-TCSPC system (Jobin Yvon, Inc., Edison, NJ, USA) with 300 μL cuvette. The FP of the sample solution was monitored by exciting the sample at 490 nm and measuring the emission at 520 nm. And slits for both the excitation and the emission were set at 5 nm. 2.3. Adenosine assay with FP aptasensors
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2.5. Procedure for detection of thrombin in human plasma samples Five normal human plasma sample collected in the citrate anticoagulated tubes was kindly provided by the No. 5 People′s Hospital (Guilin, China). Fibrinogen was removed through precipitation according to the method reported in the literature (Chen et al., 2010a, 2010b). Briefly, 0.1 mL of human plasma was quickly mixed with 1.25 mL of ammonium sulfate (2 M) and 1 mL of NaCl (0.1 M). After 4 min incubation, the mixture was centrifuged and the upper supernatant solution was collected. Then, CaCl2 (0.03 M) with 8 nM human factor Xa was then added to the plasma to promote the transformation from prothrombin to thrombin. Finally, the resulting plasma diluted 100-fold with Tris–HCl buffer (20 mM Tris–HCl, 50 mM NaCl, 10 mM Mg2 þ , and 50 mM K þ , pH 7.9), and immediately used for Tb detection by the proposed FP aptasensor. The assay procedures were the same as that of the standard Tb detection noted above.
3. Results and discussion 3.1. Principle of the amplified aptamer-based FP assay for adenosine detection By taking the advantages of NESA and GO enhancement, we present a new strategy for development of amplified FP aptasensors for detection of biomolecules. Scheme 1 depicts one amplified FP aptasensor configuration that is exemplified with detection of A. This system consists of an anti-A aptamer probe (AAA), an assistant DNA probe (FAM-labeled DNA-2), GO, and nicking endonuclease Nb.Bbvc I. The AAA probe is designed with a 10-nt extension sequence at its 5′-end. The FAM-labeled DNA-2 probe is designed to be partly complementary to the short extension sequence of the AAA probe, and also contains the cleavage site of Nb.Bbvc I. In the absence of A, the AAA probe is unable to bind with the FAM-labeled DNA-2 probe because the complementary oligonucleotides are too short to promote efficient hybridization. In this case, both AAA and FAM-labeled DNA-2 probes are adsorbed onto the GO surface, thus the FAM dye exhibits very high FP value (P) due to the extraordinarily larger volume of GO. When A is introduced to the system, the AAA probe binds the A target to form an A/aptamer complex with a duplex DNA region, and thus the enhancement of base stacking (Cai et al., 2013). This allows the A/aptamer complex to hybridize with a FAM-labeled DNA-2 probe associated with GO, resulting in another DNA duplex region containing the recognition sequence of Nb.Bbvc I. This triggers the selective enzymatic cleavage of the FAM-labeled DNA-2 probe by Nb.Bbvc I, resulting in the release of a short DNA fragment carrying the FAM dye from the GO surface and the decrease of the P value. In addition, the Nb.Bbvc I-catalyzed cleavage of the FAM-labeled DNA-2 probe releases the intact A/ aptamer complex that can then hybridize with another FAMlabeled DNA-2 probe associated with GO to initiate the cleavage of the FAM-labeled DNA-2 probe. Thus, Nb.Bbvc I recycles the
Fig. 1. FP values in the absence (black) and presence (red) of 400 nM adenosine with different sensing systems. (A) The AAA(20 nM)/DNA-2(85 nM)/GO (100 μg/mL)/Nb.BbvCI(0.5 U/μL) system; (B) the AAA(20 nM)/DNA-2(85 nM)/Nb. BbvCI (0.5 U/μL) system; (C) the AAA(20 nM)/DNA-2(85 nM) system; (D) the AAA (20 nM)/DNA-2(85 nM)/GO(100 μg/mL) system. Error bars were derived from N¼ 3 experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
A/aptamer complex, leading to the continuous cleavage of the FAM-labeled DNA-2 probes linked to GO and the release of many short DNA fragments carrying the FAM dye from GO, generating a substantial decrease of the P value, and thus achieving the target assisted dual signal amplification. Of note, the introduction of GO and Nb.Bbvc I provides significant signal amplification, which can substantially improve the detection sensitivity of the FP aptasensor compared with traditional homogeneous aptasensors. 3.2. Feasibility study To verify the feasibility of our design, the FP assay for detection of 400 nM A at different conditions was investigated, and the results obtained are shown in Fig. 1. With the AAA/DNA-2 system, the P value increased slightly upon the addition of A. However, control experiments revealed that no changes of the P value occurred when using DNA-2 only for A detection. This suggested the formation of stable A-mediated complex between the AAA probe and the FAM-labeled DNA-2 probe. When Nb.Bbvc I was introduced to the AAA/DNA-2 system, the P value decreased significantly upon the addition of A, indicating that target assisted Nb.Bbvc I-catalyzed cleavage of the FAM-labeled DNA-2 probe had taken place and generated short DNA fragments carrying the FAM dye. When the AAA/DNA-2/Nb.Bbvc I/GO system was used for A detection, the background P value was greatly enhanced to 416, which was about 4-fold higher than the situation without GO. Moreover, the ΔP value of this system for A detection is approximately 5 times higher than that of the system without GO enhancement and 36 times higher that of the system without
Scheme 1. Amplified FP analysis of adenosine using GO enhancement and nicking enzyme-aided recycling of the adenosine–aptamer complex.
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both NESA and GO enhancement. However, control experiments revealed that no apparent change of the P value occurred when the AAA/DNA-2/GO system was used for A detection. The significant enhancement of the ΔP value was attributed to the fact that GO could greatly enhance the background P value and short fluorescent DNA fragments with faster rotation were released from GO by target assisted Nb.Bbvc I-catalyzed cyclic cleavage of the FAM-labeled DNA-2 probe linked to GO. These results demonstrated that the introduction of GO and Nb.Bbvc I could significantly amplify the reporting signal for the FP aptasensor.
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GO. Thus, the background FP value would be low. On the other hand, too much of GO would inhibit the release of the cleaved products from GO and result in a poor reporting signal. Thus, it is necessary to optimize the amount of GO to give the largest ΔP enhancement. Fig. S3 shows the effects of different concentrations of GO on the ΔP value. As the concentration of GO increased, the ΔP value increased gradually upon the addition of A. The largest ΔP value was obtained when 100 μg/mL GO was employed. Further increasing GO concentration, the ΔP value decreased, which might be attributed to the inhibition of the release of the cleaved products from GO by high concentration of GO.
3.3. Optimization of assay conditions 3.4. Adenosine assay For such FP amplifying design, the complementary sequence of the AAA probe to the assistant DNA probe is a critical factor. On the one hand, the FP signal amplification promoted only in the presence of A as we designed, so if the complementary bases is too long, it may initiate the FP signal amplification both in the presence and absence of A. On the other hand, too short complementary bases would not initiate the FP signal amplification even in presence of A. Therefore, it is essential to optimize the complementary bases between the AAA probe and the assistant DNA probe. Our AAA probe was designed with a 10-nt extension sequence at its 5′-end, four assistant DNA probes that contains different complementary bases to the AAA probe were tested. Fig. S2 shows the effects of four assistant DNA probes with 7–10 complementary bases of the AAA probe on the P value of the amplified sensing system in the absence and presence of A. As can be seen, the DNA-2 probe with 8-nt complementary bases provided the best signal to background ratio. This was attributed to the fact that the DNA-2 probe with 8-nt complementary bases could stabilize the hybridization between the AAA probe and the assistant DNA probe and then triggered an effective NESA process only in the presence of A. Therefore, the DNA-2 probe was selected as the optimal assistant DNA probe for the following assays. In addition, the amount of GO also significantly affected the reporting FP signal. On one hand, if the amount of GO is too small, the FAMlabeled DNA probe will not be adsorbed enough on the surface of
The proposed amplified FP aptasensor for A detection is highly sensitive. Fig. 2, curve a, shows the FP responses of the proposed amplified sensing system upon analyzing different concentrations of A, for a fixed reaction time-interval of 1 h. With increasing of the A concentration, the ΔP value increased gradually, consistent with the enhanced cleavage of the FAM-labeled DNA-2 probe linked to GO by Nb.Bbvc I-recycled the A/aptamer complex and the release of more amounts of short DNA fragments carrying the FAM dye from GO. Interestingly, as shown in Fig. 2, inset, the plots of the ΔP value versus A concentration in the range of 4 pM–10 μM showed good linear relationships (R2 ¼0.9981). This amplified FP aptasensor allowed the detection of A with a detection limit corresponding to 2.0 pM, which was at least four orders of magnitude lower than that of traditional homogeneous aptasensors, and two orders of magnitude lower than that of the reported amplified aptamer-based homogeneous assays (Table S1). In contrast, when the FP aptasensor without GO enhancement was used, a sensitivity that corresponds to an A concentration of 0.2 nM was obtained (Fig. 2, curve b); when the FP aptasensor without NESA and GO enhancement was used, a sensitivity that corresponds to an A concentration of 200 nM was obtained (Fig. 2, curve c). The substantial sensitivity improvement of this amplified FP aptasensor was attributed to the reasons as follows: 1) GO with large volume could greatly enhance the background P value when the
Fig. 2. Plots of FP changes as a function of adenosine concentrations using different sensing systems. (a) The AAA(20 nM)/DNA-2(85 nM)/GO(100 μg/mL)/ Nb.BbvCI(0.5 U/μL) system; (b) the AAA(20 nM)/DNA-2(85 nM)/Nb.BbvCI (0.5 U/μL) system; (c) the AAA(20 nM)/DNA-2(85 nM) system. Inset. The derived calibration curve corresponding to the △P value of the AAA/DNA-2/ GO/Nb.BbvCI system. Error bars were derived from N ¼3 experiments.
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FAM-labeled DNA-1 probe was adsorbed onto GO; 2) the A target assisted Nb.Bbvc I-catalyzed cleavage of the FAM-labeled DNA-1 probe resulted in the release of many short DNA fragments carrying the FAM dye with faster rotation. This amplified FP aptasensor for A detection was also specific. To evaluate this property, we tested the response of the assay to the target A and several analogue molecules: cytidine (C), guanosine (G), and thymine (T). It was found that the specific target A led to an increase in the ΔP value, and no obvious change of the ΔP value was observed in the assay for all analogue molecules as compared to the background (Fig. S4). These results clearly demonstrated the high specificity of our amplified FP aptasensor. In addition, the amplified FP aptasensor also exhibited acceptable precision, and the variation coefficients (CVs) for five repetitive measurements of 40 pM, 8 nM, and 0.8 μM A were 1.8–5.4%, which were comparable to the reported methods (Cai et al., 2013; Chen et al., 2008). 3.5. Thrombin detection To illustrate the generality of our strategy, we applied this strategy to develop another amplified FP aptasensor for Tb detection (Scheme 2). Thrombin (Tb) was used as a model analyte. This sensing platform involves the use of the aptamer subunits approach. The two anti-Tb aptamer subunits ATA-1 and ATA-2 are used, and ATA-1 includes a 10-nt extension sequence at its 5′end that is designed to be partly complementary to the FAMlabeled DNA-2 probe. The aptamer subunits and the FAM-labeled DNA-2 probe can coexist stably in the absence of Tb, and they are adsorbed onto GO, thus the FAM dye exhibits very high P value. However, the addition of Tb results in the assembly of two aptamer subunits into a Tb/aptamer complex with a duplex region, which promotes the hybridization between the FAM-labeled DNA2 probe and the Tb/aptamer complex to form a duplex domain containing the cleavage site of Nb.Bbvc I. This triggers the Nb.Bbvc I-catalyzed selective cleavage of the FAM-labeled DNA-2 probe, resulting in the removal of the FAM dye from GO, the release of the Tb/aptamer complex and the decrease of the P value. The released Tb/aptamer complex can then hybridize with another FAMlabeled DNA-2 probe linked to GO, and the cycle starts anew, which leads to significant amplification of the reporting signal. By monitoring the decrease in the P value, we could detect Tb with very high sensitivity. Firstly, the key factors affecting the amplified FP assay for Tb detection were optimized (Figs. S5 and S6). After that, the ability of the amplified system for Tb detection was demonstrated. As shown in Fig. 3, curve a, the amplified FP aptasensor based on NESA and GO enhancement enabled the detection of Tb with a detection limit of 1 fM, which was three orders of magnitude lower than that of the FP aptasensor without GO enhancement (0.1 pM) (Fig. 3, curve b), and five orders of magnitude lower than that of the FP aptasensor without NESA and GO enhancement (1 nM) (Fig. 3, curve c). Meanwhile, the detection limit for Tb in this work was also four orders of magnitude than that of
traditional homogeneous aptasensors and two orders of magnitude lower than that of the reported amplified aptamer-based homogeneous assays (Table S1). The sensitivity improvement of this amplified FP aptasensor for Tb detection was attributed to the fact that GO associated with the FAM-labeled DNA-2 probe could greatly enhanced the background P value, and many short DNA fragments carrying the FAM dye with faster rotation were released from GO by Tb assisted Nb.Bbvc I-catalyzed DNA cleavage reaction. The results also showed good linear relationships (R2 ¼0.9966) between the ΔP value and Tb concentration in the range of 2 fM– 200 nM (Fig. 3, inset). This amplified FP aptasensor also showed high specificity toward Tb over other non-specific proteins which were co-existing in biological samples, such as IgG, IgE, HSA and AFP (Fig. S7). Moreover, this amplified FP aptasensor also exhibited acceptable precision, and the CVs for five repetitive measurements of 20 fM, 2 pM, and 2 nM Tb were 2.2–5.6%, which were comparable to the reported methods (Yuan et al., 2011; Huang et al., 2013). 3.6. Detection of thrombin in human plasma To evaluate the application of the amplified FP aptasensor to real sample, the determination of Tb in five human plasma samples was conducted by the present method and compared with those obtained by the traditional ELISA method. As shown in Table 1, the assay results of Tb in human plasma by the amplified FP aptasensor were in an acceptable agreement with those of the ELISA method, and were also consistent with the reported levels (Chen et al., 2010a, 2010b; Wang et al., 2011). Moreover, the method also revealed the good recovery rates of standard addition from 94.2% to 105.2%. In addition, the assay precision was also evaluated by repeatedly analyzing each human plasma sample five times within a working day, and the relative standard deviations for assays were in the range of 3.4% to 4.8%. These results indicated that the proposed amplified FP aptasensor could be acceptable for quantitative assays performed in biological samples.
4. Conclusions In summary, we have developed two different configurations for developing new amplified FP aptasensors based on both NESA and GO enhancement. By the appropriate design of the aptamer probe or aptamer subunits, and a fluorophore- labeled assistant DNA probe, these configurations can be effectively applied for highly sensitive and selective detection of A or Tb (as model analytes). Furthermore, the suitability of the amplified FP aptasensor for complex biological sample analysis has also been demonstrated. These assays are quite simple and convenient, needing only mixing the GO-functionalized oligonucleotide probes, the nicking endonuclease, and the target molecule in homogeneous solution, and not requiring separation and troublesome procedures. Moreover, the sensitivity of these amplified FP
Scheme 2. Amplified FP sensing of thrombin using two aptamer subunits and nicking enzyme-catalyzed regeneration of the thrombin-aptamer complex.
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Fig. 3. Plots of FP changes as a function of thrombin concentrations using different sensing systems. (a) The ATA-1(20 nM)/ATA-2(20 nM)/DNA-2(85 nM)/GO(100 μg/mL)/Nb. BbvCI (0.5 U/μL) system; (b) the ATA-1(20 nM)/ATA-2(20 nM)/DNA-2(85 nM)/Nb.BbvCI (0.5 U/μL) system; (c) the ATA-1(20 nM)/ATA-2 (20 nM)/DNA-2(85 nM) system. Inset. The derived calibration curve corresponding to the ΔP value of the ATA-1/ATA-2/DNA-2/GO/Nb.BbvCI system. Error bars were derived from N ¼ 3 experiments. Table 1 Determination of thrombin in five human plasma samples. Sample
Present method (nM)
ELISA (nM)
Relative deviation (%)
1 2 3 4 5
86.3 102.4 119.2 103.6 96.3
89.6 97.5 110.8 108.7 103.3
3.7 5.0 7.6 4.7 6.8
aptasensors is at least four orders of magnitude higher than that of traditional unamplified aptamer-based homogeneous assays. Additionally, these approaches may also be extended to other target molecules detection by simply switching the corresponding aptamer probes or aptamer subunits. Finally, these assays can be easily carried out in 96- or 384-well plates, rendering it suitable for routine high-throughput applications. Thus, the proposed amplified FP aptasensors can be expected to provide sensitive and general platforms for amplified analysis of target molecules.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 21305021 and 21175030), the National Basic Research Program of China (No. 2012CB723501), and the Natural Science Foundations of Guangxi Province (Nos. 2012GXNSFDA385001, 2013GXNSFBA019038, and 2013GXNS FBA019044) as well as BAGUI Scholar Program and the Project of Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Education of China (CMEMR2012-A19 and CMEMR2013-C06).
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.036.
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