Bivalent Aptasensor Based on Silver-Enhanced Fluorescence

Subscriber access provided by Van Pelt and Opie Library

Article

Bivalent aptasensor based on silver enhanced fluorescence polarization for rapid detection of lactoferrin in milk Zhu Chen, Hui Li, Wenchao Jia, Xiaohui Liu, Zhoumin Li, Fang Wen, Nan Zheng, Jindou Jiang, and Danke Xu Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 4, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Bivalent aptasensor based on silver enhanced fluorescence polarization for rapid detection of lactoferrin in milk Zhu Chena, Hui Li*a, Wenchao Jiaa, Xiaohui Liua, Zhoumin Lia, Fang Wenb, Nan Zhengb, Jindou Jiangc, Danke Xu*a a

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210046, China b Ministry of Agriculture-Key Laboratory of Quality & Safety Control for Milk and Dairy Products, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China c Dairy Quality Supervision and Testing Center, Ministry of Agriculture, Harbin 150090, China Corresponding Author Tel/Fax (+) 00862583595835 E-mail: [email protected], [email protected]

Abstract Here we report a novel type of bivalent aptasensor based on silver enhanced fluorescence polarization (FP) for detection of lactoferrin (Lac) in milk powder with high sensitivity and specificity. The novel two split aptamers were gotten from the aptamer reported in our previous SELEX (systematic evolution of ligands by exponential enrichment) selection and their minimal structural units were optimized based on their affinity and specificity. Also, dual binding sites of split aptamers were verified. The bivalent aptamers were modified to be linked with signal molecule fluorescein isothiocyanate (FITC) and enhancer sliver decahedral nanoparticles (Ag10NPs) respectively. The split aptamers could bind to different sites of Lac and assembled into a split-aptamers-target complex, narrowing the distance between Ag10NPs and FITC dye. As a result, Ag10NPs could produce mass-augment and metal-enhanced fluorescence(MEF) effect. In general, ternary amplification based on Ag10NPs, split aptamers and MEF effect all contributed to the significant increase of FP values. It was proved that the sensitivity of this assay was about three orders of magnitude over traditional aptamer-based homogeneous assays with detection limit of 1.25 pM. Furthermore, this design was examined by actual milk powder with rapid and high throughout detection. Keywords Silver decahedral nanoparticles; Aptamers; Lactoferrin; Fluorescence polarization; Bivalent aptasensor; metal-enhanced fluorescence effect

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Introduction Aptamers are functionally single-stranded DNA or RNA(10–100 nucleotides) that can bind to a variety of target molecules including small molecules, virus, proteins, peptides and even cells with distinctive sensitivity and specificity.1,2 Aptamers are generally obtained by a vitro SELEX progress from a large random sequence pool and show strong affinity with the target molecule even over antibodies.3,4 Compared with naturally occurring or artificial receptors, for example, antibodies or molecularly imprinted polymers, aptamers offer several outstanding properties in practical application such as simple synthesis, flexible modification, good stability, low cost and so on.5-7 In the presence of target, aptamers significantly transforms into hairpins, stem-loops, G-quadruplexes and so on, to bind target molecule.8 Up to date, hundreds kinds of aptamers have been reported.9 To circumvent the drawback of steric hindrance, aptasensors based on split aptamers were designed to improve the detective ability which exhibited well enhanced effect by increasing signal-to-background ratio.10-13 In the past years, some efforts have been initiated to combine split aptamers with versatile materials for the design of various electrochemical or fluorescent biosensors. Briefly, fluorescent biosensors can be generally categorized into following groups: signal-on14-15 or signal-off16-17 fluorescent methods, colorimetric methods, 18-19 chemiluminescence methods20-24 and other methods25.6 On the whole, these biosensors were all based on sandwich assay and split aptamers were simultaneously used to capture target and generate signal. This feature exhibits some difference from traditional sandwich assays on the number of recognition elements against targets as all split aptamers reported were self-assembled into the intact aptamer to specifically interact with target. Regarding proteins with larger molecular weight, FP is an effective method due to its sensitivity of the rate of rotation depending on the target-induced structural or conformational change.26,27 Therefore, there are several advantages such as its homogeneous reaction, accuracy, simplicity, speed and automated high-throughput capability.28-29 Direct detection of target by FP method stands inferior status compared with other detection assays as FP method generates limited signal and substantial background.30-31 Signal amplified measurements including structure-switching, 32 mass-augmented33 and target-induced displacement34 have been developed for quantitative analysis of target molecules. For example, Jiang33 invented a mass-augmented FP method for both Hg2+ and cysteine detection and first introduced AgNPs to FP system. The enhancement of FP signal was due to the effect of QD and AgNPs on mass augment. Only two reported assay of dual amplified mechanism were both developed by Huang. Huang35 first conceived a FP aptasensor based on nicking enzyme and grapheme oxide. Their enhanced mechanism was ascribed to the mass-augmented effect of GO and enzyme reactions. The other work36 was based on cascade strand-displacement and polystyrene nanoparticles(PS NPs) for the detection of proteins. Target-induced cascade strand displacement and PS NPs mass-augmented amplification were both utilized to enhance FP signal. Despite of the fact that several aptamer-based amplified FP aptasensors have achieved preferable detection results, it is not enough to satisfy the meet of practical detection due to the lack of more amplified methods. And, to the best of our knowledge, amplified strategies combining MEF effect of Ag10NPs with FP assay haven’t been developed. Herein, we developed a novel FP aptasensor for Lac detection based on bivalent aptamers and Ag10NPs. To fabricate the ternary amplified aptasensor, the optimal aptamer Lac-A4 screened in previous report37 was used for this study. The sequence of 50-mer bivalent aptemer was


Page 2 of 16

Page 3 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


innovatively split into two fragments, one was assembled on the surface of Ag10NPs and the other was labeled with FITC dye. It was proved that not only bivalent aptamers but also MEF effect could tremendously improve sensitivity of FP. The proposed aptasensor showed highly sensitive response to Lac and the feasibility for actual samples was also proved. The presented aptasensor provids a new approach for rapid detection of the Lac in milk powder.

EXPERIMENT SECTION Materials and Regents. All oligonucleotides were synthesized by Sangon Biotech Co. Ltd (Shanghai, China), purified with high performance liquid chromatography and subsequently confirmed by NanoDrop 2000. All sequences of split aptamers were listed in Table 1.The aptamer Lac-A4, was selected and reported in our previous work, denoted as PMM-SELEX.37 Lactoferrin (Lac), α-lactalbumin (α-Lac), β-lactoglobulin (β-Lac), casein(Cas) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. Six kinds of milk powders including MeadJohnson Enfinitas (with Lac), MeadJohnson Enfamil (without Lac), Carecoming Lactoferrin (with Lac), Carecoming Whole(without Lac), Beingmate Love (with Lac) and Beingmate Champion Baby (without Lac) were purchased from shopping mall respectively. Phosphate buffers were used to dilute aptamers and milk powders in the experiment: 1×PBS（137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L, Na2HPO4.12H2O, 2 mmol/L KH2PO4）, 1× PBSM (1× PBS, 1 mmol/L MgCl2 ), pH 7.4). Mg2+ was used to keep the three-dimensional structure of aptamers when binding to proteins. Apparatus and Synthesis. The FP signals were measured on BioTek (Synergy H1, USA). Excitation wavelength was set at 488±20 nm and emission wavelength was recorded at 520±20 nm. Ag10NPs involved in this work was synthesized, purified and characterized by the assay previously reported by Li.38 The preparation and characterization of Ag10NPs were shown in Figure S1（Supporting Information）with 50.3±4.0 nm diameter. Based on our previous experience in using Ag10NPs39-41, the synthetic samples can be placed for two months at room temperature. The ultraviolet-visible (UV-Vis) spectra of Ag10NPs at 0 day, 1 days, 3 days, 5 days, 7 days, 10 days, 15 days were offered in Figure S2（Supporting Information）. The maximum peaks of UV-Vis spectra were 480 nm which only shifted slightly during 15 days. The difference could be ignored and the stability of Ag10NPs was acceptable within a 15-days period. The concentration of Ag10NPs was approximately calculated as 0.56 nM by treating its shape as sphericity. All other reagents contained in this work were of analytical grade, unless otherwise stated. Coefficient effect of two parts split by a aptamer. The website of IDT was used to simulate and edit secondary structure of aptamers. One aptamer was picked out from our selected and reported aptamers for further experiment (denoted as Lac-A4, ∆G = -11.21kcal/mol).37 The synthesized aptamers were adequately diluted with 1×PBS to form 100 µM of stock solutions and stored at 4℃. Briefly, FITC labeled aptamers(10 µL, 250 nM ) were separately mixed with proteins (100 µL, 5 µg/mL) and incubated without light for 15 minutes at 37℃ with rapid shaking. Then FP value were measured on BioTek. All experiments were repeated in triplicate. To amplify the FP value of this assay, the aptamer was split into different parts as shown in Table 1 and Figure 1(a). Two split aptamers (10 µL, 250 nM) were mixed with proteins (90 µL, 1 µg/mL)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and incubated without light for 15 minutes at 37℃with rapid shaking. Control experiments were also carried out with the intact aptamer Lac A4 to assess the amplified effect on FP value. Signal amplified design combining spilt aptamer with Ag10NPs. The principle of our assay bridging split aptamers with Ag10NPs for Lac measurement was schematically demonstrated in scheme 1. Briefly, one split aptamer was self-assembled on the surface of Ag10NPs through sulfydryl group. Briefly, 2 mL Ag10NPs was mixed with DNA-2(70 µL, 10 µM), NaCl (64 µL, 2 M), Tween 20(10 µL). Subsequently, the whole suspension was incubated at 37 ℃for 2 h, vibrated vigorously and then centrifuged twice to remove the unmodified DNA chains. Next, two modified split aptamers (10 µL, 250 nM) were mixed with proteins(90 µL, 1 µg/mL) and incubated without light for 15 minutes at 37℃ with rapid shaking. Actual detection of milk powders. The content of Lac in milk powders available in the market ranges from 10 µg/100 g to 500 µg/100 g. Three representative copies(38 µg/100 g, 100 µg/100 g and 450 µg/100 g respectively) of milk powders were used to test the performance under complicated conditions. Primarily, milk powders were dissolved and diluted to a befitting level and filtered through 0.45 µm membranes for later use. Six kinds of milk powders were divided into three control groups in accordance with their brand, one having Lac while the other not. Then two modified split aptamers(10 µL, 250 nM) were mixed with different diluted milk powders , incubated without light for 15 minutes at 37℃ under rapid shaking frequently and transferred into the individual wells and processed by above steps. Finally, the data of FP was collected and analyzed to evaluate the effectiveness of this assay. RESULTS AND DISCUSSION Signal amplified design using split aptamers. The split aptamers can be generated by dividing a well-defined aptamer into two fragments. The difficulty of engineering aptamers into split aptamers likely arises from the fact that many aptamers have a hairpin or hairpin-like architecture, and the target often binds to nucleobases in the stem or loop region of the hairpin.5 Then the unpredictability of such structural alteration may generate false positive or nonspecific signals, hindering its further application in actual detection.5 The split aptamers with inappropriate artificial truncation often perturb its secondary structure, thus weakening the specific binding to the target. The part that is not necessary for binding to targets can be used as a probable split site. 6 Overall, Our aptamer consists of three stem-loops (stem-loop 1, 2 and 3). Considering reported studies on binding sites of aptamers, the two smaller stem-loops (stem-loop 1 and 2) were preliminarily envisioned to participate in the recognition of Lac, while the larger one was considered to be only acted as a framework. To evaluate the assembled properties of each split aptamers, aptamer Lac-A4 was divided into two pieces at different sections as sensing probes, one was labeled with FITC and the other was label-free. In this way, we obtained three groups of split aptamers as shown in Figure 1(a): DNA-1 （15 bases） and DNA-2 (35 bases), DNA-3 (25 bases) and DNA-4 (25 bases), DNA-5 (35 bases) and DNA-6 (15 bases). DNA-1, DNA-3 and DNA-5 were modified with FITC at its 5′end. Then the FP values of three groups of split aptamers were tested and the results were shown in Figure 1(b). In the absence of target protein, three pairs of split aptamers exhibited relatively low


Page 4 of 16

Page 5 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


background of FP value due to their smaller size. However, upon recognizing and binding with protein, FP values increased greatly as the structure of split aptamer favored the formation of a stable split-aptamers-target complex, inducing the reduction of FITC’s mobility, thus causing the increase of FP signal. The split aptamers DNA-3 and DNA-4 showed competitive advantages over other split aptamers and Lac-A4. As DNA-3 and DAN-4 retained the original structure of two smaller loop while the larger one was damaged, validating the effectiveness of stem-loop 1 and stem-loop 2 for binding with proteins. The other two groups, in which two smaller stem-loops were damaged respectively, also showed marked change compared to the background but were inferior to Lac-A4. Based on these facts, we inferred that two smaller stem-loops played a key role in targeting with Lac. And we would test the function of two smaller stem-loops in targeting protein afterwards. DNA-3 and DNA-4 was chose as two split aptamers and their structures were further optimized to increase the FP values. Structural optimization of the split aptamer. The structure of two split aptamers were further optimized to find out their minimal structure unit for target binding. All DNA probes used were illustrated in Figure 2(a). Firstly, seven paired bases, the hybridization parts on the bottom of two split aptamers were tested to figure out if these paired bases were necessary for binding with Lac. The procedure of our strategy was shown in Figure 2(a). 3, 5 and 7 paired bases were respectively removed to study the effect of paired bases number on FP value (denoted respectively as DNA-7 and DNA-8, DNA-9 and DNA-10, DNA-11 and DNA-12). Figure 2(b) showed that along with the decreased number of paired bases, FP value increased gradually and DNA-11, DNA-12 which had no paired bases showed the strongest signal. Overall, the decreased number of paired bases meant less hybridization rate of two split aptamers. So these data could clearly prove that seven paired bases on the bottom of two split aptamers were useless and DNA-11 ,DNA-12 were chosen for following study. Next, we further verified the importance of two smaller stem-loops. The loop structure of stem-loop 1 and stem-loop 2 were destroyed respectively (denoted as DNA-13 and DNA-14). When the loop structure of signaler was destroyed, DAN-13, obviously reduced signal, only a little higher than background, was observed in Figure 3(b). It’s no doubt that the loop part of stem-loop 1 participated in the recognition of Lac. The damage of the loop structure made it fail to bind with Lac, thus DNA-13 was in a individual state. For the same reason, when the loop of stem-loop 2 was destroyed, apparent decreased FP value was also observed, obviously weaker than that of DNA-11 and DNA-12. The observed signal was ascribed to the specific binding of stem-loop 2 to Lac. From these results, we could conclude that the loop region of stem-loop 1 and stem-loop 2 were important for targeting binding. To further find out the binding mechanism between two split aptamer and Lac, the function of each split aptamer was examined. For certain, the binding mechanism of split aptamer would be different from the existing one as they didn’t reassemble into the intact one but both separately recognize Lac. To test if the two split aptamer bound with protein at the same site or different sites, we designed and modified the split aptamers just as shown in Figure 3(a). The two optimized split aptamers, DNA-11 and DNA-15, were both labeled with FITC. Equimolar DNA-11 and DNA-15 were processed in this assay respectively or simultaneously. Data recorded in Figure 3(b) showed that the signal generated by the mixture were higher than their individual, almost 1.6 times of independent signals, further proving the speculation of dual recognition sites. In this case, if two



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 16

probes bound at the same site, the addition of any probe only caused competition balance and would not change the FP value too much. Theoretical signal of the mixture should be between two individual one. So this experiment could serve as the sufficient proof of different binding sites to Lac. Although many scholars used sandwich method and pre-existing split aptamers to construct analytical methods. In this study, we originally split the screened aptamer Lac-A437to find out the novel split aptamers with high affinity for Lac. Moreover, the split aptamers were totally new aptamers with different binding sites towards Lac. The method design was not based on the whole aptamer structure self-assembled by split ones, but on the dual binding features of two split ones. We actually found that both split aptamers could bind to Lac for which we considered it different from the reported mechanism. Besides, Lac is a nonheme iron-binding glycoprotein strongly expressed in bovine and human milk and it plays important functions during infancy. We aimed to establish a rapid and sensitive method for detection of active Lac in milk. MEF enhanced effect of Ag10NPs. Based on above analysis, despite that our assay showed preferable sensitivity against Lac, novel enhanced method was urgent to be invented according to split aptamer’s conformational change or structural transformation upon interaction with the target. By taking the advantage of the MEF effect of Ag10NPs on fluorescent dye, we presented a new strategy for the development of FP amplifier. Briefly, DNA-12 was lengthened by sulfydryl group modified10-mer polyA and then assembled with Ag10NPs (denoted as capture probe). DNA-11 was labeled with FITC dye at its 5′end, denoted as signal probe. The 10-mer polyA between 3′end of DNA-12 and Ag10NPs was designed as a spacer to make aptamer far from the surface of Ag10NPs that could maintain the conformation of probes for recognition of target. Then the Ag10NPs modified split aptamer and FITC labeled one were designed to interact with the target collectively in sandwich assay.The amplified mechanism of MEF effect on FP was specified next. Generally, the observed polarization will depend not only on the rotational rates but also the excited state lifetime. For simplicity, the polarization can be written in a rearranged form of the well-known Perrin Equation （1）.42

（1）

In this equation, P is the observed polarization, P0 is the limiting polarization that cam be treated as a constant, τ is the excited state lifetime, and φ is the rotational correlation time which is related to the rotational relaxation time (ρ) by the expression ρ= 3φ. So FP is in verse proportion to τ while in direct proportion to ρ. The effective MEF effect made the relaxation time of FITC longer than its individual state as it was more difficult to overcome the interaction force before reaching equilibrium states (ρ↑), and also shortened the excited state lifetime (τ↓) of the dye according to the radiative decay theory, thus both causing the increase of FP value (P↑). So MEF effect could enhance the FP signal by extending relaxation time of molecular motion and shortening excited state lifetime. First, we explored the optimized ratio of probes and protein. Aptamers and split aptamers stock solutions were taken out from the fridge and placed to room temperature. With the concentration of Lac set as 5 µg/mL , different concentrations of 500, 250, 125 or 62.5 nM signal probe were added separately into the system. From figure 4 (a), when the concentration of signal probe was 250 nM, the ∆FP value was highest, so 250 nM was chosen for the next experiments.


Page 7 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Subsequently, different length of DNA chains on Ag10NP were evaluated to find out the most suitable one for silver-enhanced FP aptamer sensor. Four paired DNA chains were modified with Ag10NPs or FITC simultaneously and their FP signal were recorded in Figure 4 (b). Obviously, DNA-9 and DNA-10 which had two paired bases were the best. This DNA length ensured the appropriate space between Ag10NPs and probes for easy recognition of Lac, causing the MEF effect on FITC. The increase number of paired bases would result in unexpected hybridization with capture probes. So DNA-3 and DNA-4, DNA-7 and DNA-8 exhibited considerable background. At the same time, in order to further define the optimal conditions for FP detection, the effect of the concentration of Ag10NPs was evaluated. DNA-9 and DNA-10 were modified and used as signal probe and capture probe. The pre- synthetic Ag10NPs were concentrated or diluted to different fold: 12, 25, 50 and 100 DNA-12 chains per Ag10NP respectively and then were modified as before. The final FP singal was recorded in Figure 5 (a). Along with the increase of DNA-10 chains, the background signal kept increasing all the time. Meanwhile, FP value of the probes-Lac system also increased and 50 DNA-10 chains per Ag10NP showed the highest FP signal. This number of DNA-10 chains modification ensured the proper distribution density of DNA-10 chains on the surface of Ag10NP. So 50 DNA-10 chains per Ag10NP was chose as optimal condition and then used for further study in this assay. Finally, in order to verify the feasibility of our designed assay, signal probe and capture probe, Lac-A4, DNA-9 and DNA-10 were used as models, then the FP value was investigated in different sensing systems for the detection of 1 µg/mL Lac. Results were shown in Figure 5 (b). With the use of capture probe only, FP value changed slightly upon the addition of Lac for the reason that capture probe was fluorescent-free. With the use of signal probe only, FP value increased notably upon the addition of Lac, suggesting the recognitive function of stem-loop 1. Then, when the Ag10NPs assistant capture probe and signal probe were introduced for Lac detection, FP value sharply increased compared with that of background, much higher than that of Lac-A4 or DNA-9 and DNA-10. These results demonstrated that the import of Ag10NPs could tremendously amplify the signal of split aptamer sensor. And in the same way, the sensor based on DNA-9 and DNA-10 provided increased FP signal than Lac-A4 maybe due to their decreased steric hindrance. Overall, in the abscebce of Lac, capture probe and signaler probe were independent in the absence of Lac and Ag10NPs had no MEF effect on FITC. However, in the presence of Lac, the target assembled two probes into a probes-aptamer complex and then induced the MEF effect of Ag10NPs. So we concluded that this amplified FP aptasensor contained ternary amplifier: mass-augmented, split aptamers enlarged and MEF-enhanced amplification. Besides, the dual binding properties was similar to bivalent antibody which has two binding sites for antigens. Then we denoted it as bivalent aptamers and we split it into two recogntive fragments. The Kd of the Lac-A4 (a), DNA-11(b), DNA-15(c) and the random chains with the same chain length 52 bases(d), 18 bases (e) were shown in Figure S3（Supporting Information. All three DNA chains: DNA-11, DNA-15, Lac-A4, showed comparable affinity with Lac, respectively quantified as 8.86 ± 1.72 nM, 28.78 ± 7.20 nM and 4.97 ± 0.46 nM. Considering the same bases of DNA-11 and DNA-15, the difference of binding affinity can be inferred from figure 3(b) in which DNA-11 showed certain higher FP signal than that of DNA-15. Control chains showed no affinity towards Lac even at the concentration of 1600 nM. Besides, the ∆G of three chains: Lac-A4, DNA-11, DNA-15, were respectively denoted as-11.21 kcal/mol, -5.93 kcal/mol and -2.27 kcal/mol



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

corresponding to their Kds, also revealing their stability in short DNA chains. For capture probes, the smaller stem-loop of DNA-15 ensured better formation of Ag10NPs probes- target complex with smaller steric hindrance. So DNA-15 was chosen as capture probes. Compared with previously reported mechanism of split aptamers, this bivalent one had greater application value to construct well-functioned aptasensor. Split aptamers reported self-assembled into an intact aptamer in the presence of target and failed to interact with the target individually. Conversely, in this assay, both split aptamers participated in the recognition of Lac independently and bound to different sites of Lac. This specificity can be used for dual channel signal output, thus more convenient for monitoring the combining procedure of sandwich aptasensor. Besides, this discovery opened a door for the study of bivalent aptamer, thus taking a step forward on the way of functionally displaying antibodies. In short, we originally achieved following results in this study. Firstly, although the method of split aptamers has been used many times, we explored the way of splitting the whole aptamers into two new subunits and its potential applications. Secondly, split aptamers were used to study the binding mechanism of aptamers to Lac, laying solid foundation for the construction of ultrasensitive aptasensors. Finally, we obtained two new aptamers for the detection of Lac with comparable affinity as the whole aptamer Lac-A4. To avoid yielding false-positive or nonspecific signals, four negative proteins, whose concentrations were 50 µg/mL were used as controls to test the specificity of this aptasensor. The concentration ratio between negative proteins and Lac was set referred to their actual ratio in milk powders. Figure 6(a) showed the superb specificity of Lac over other negative proteins as a sharp increase in FP value while no apparent change was observed for all negative proteins, thus identifying the target specificity of this sensing system. Subsequently, to evaluate the sensitivity of amplified FP aptasensor combining bivalent aptamer with Ag10NPs enhancement for Lac detection, aliquots of various concentrations of Lac were prepared and then activated in probe solutions respectively. capture probe, signaler probe, and Lac assembled into a complex with high weight, thus significantly increasing the FP value of complex. As the concentration of Lac was higher, the relative FP value increased gradually, consistent with the generation of more probes-Lac complexes and MEF effect. Importantly, as shown in Figure 6 (b) and 6(c), there was an excellent linear relationship between ∆FP and Log C. For amplified aptasensor, the plots of the relative ∆FP value versus the logarithm of Lac concentrations showed a good linear relationship in the range of 0.2 ng/mL-25µg/mL, R2 = 0.998. Based on a single-to-noise ratio >3, the detection limit for Lac was estimated to be 0.1 ng/mL (1.25 pM), which was about 3 orders of magnitude lower than that of traditional unamplified aptamer-based homogeneous assays (For Lac-A4, the linear range was from 195 ng/mL to 25 µg/mL with the detection limit 98 ng/mL, 1.22 nM, R2 = 0.993). Outstanding achievements in this method may be ascribed to the dual recognition sites, mass augment, relatively low background of split aptamer and MEF effect of Ag10NPs. Practical applicability of the designed aptasensor. To ultimately evaluate the general applicability of Ag10NPs-assistant aptasensor, the current approach was tested by three groups of milk powered samples. Three non-Lac contained copies of milk samples were used as control and their experiment data was treated as background signal. Firstly, MeadJohnson Enfinitas milk powered was used to study the optimal dilution factor. 12.5 ng/mL, 5 ng/mL, 2 ng/mL and 0.8 ng/mL Lac were added into diluted samples respectively and FP values were analyzed by calculating their recovery rates. On the basis of Table 2, 100-fold dilution was used to be the


Page 8 of 16

Page 9 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


measurement condition due to its good recovery rates of standard addition between 97.5 % and 103.3 %. These results indicated that the proposed amplified FP aptasensor could be acceptable for quantitative assay of Lac in complicated samples. Secondly, standard addition experiments were also performed with these milk powered samples and conducted under the optimal experiment conditions to validate the assay. Results were recorded respectively in Figure 7 (a) , Figure 7 (b) and Figure 7 (c). This method provided very good linear results in actual detection of samples and their linear range was respectively fitted as 12 ng/mL-25 µg/mL, R2=0.996; 3 ng/mL-25 µg/mL, R2=0.993; 0.8 ng/mL-25µg/mL (R2=0.996), revealing some difference with that of buffer solutions. Finally, authentic Lac-contained samples were detected by this assay and the resulting data was substituted into the linear formula to calculate the theoretical concentrations of samples. Results of theoretical concentrations were in an acceptable agreement with labeled concentrations and the relative standard deviations for this assay were in a reasonable range. Results in Figure 7 (d) further validated the feasibility of proposed FP aptasensor in actual samples.

CONCLUSION In summary, the feasibility of a highly sensitive FP aptasensor based on Ag10NPs enhancement, split aptamers enlargement and MEF amplification have been demonstrated. This amplified strategy employed dual recognition of the two split parts and MEF effect of Ag10NPs on FITC. The developed assay showed highly sensitive response to Lac with the detection limit of 1.56 pM, which was superior than the majority of previously reported methods for Lac detection. Moreover, this strategy was further carried out to detect milk powders with high recoveries and low interference. The presented approach substantially broadened the perspective for FP amplified probes.

AUTHOR INFORMATION Corresponding Author *Danke Xu, Hui Li, Tel/Fax: (+) 00862589685835. E-mail: [email protected]. [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS Financial support was provided by National Natural Foundation of China (Grant Nos. 21405077, 21227009, 21475060), Natural Science Foundation of Jiangsu Province (BK20140591), Special Fund for Agro-scientific research in the Public interest(201403071) and the National Science Fund for Creative Research Groups(21121091).

ASSOCIATED CONTENT *S Supporting Information Detailed information of the synthesis process, TEM images, UV-Vis spectra characterization of Ag10NPs, Kds of split aptamers and Lac-A4, the Linear relationship of non- Ag10NPs assistant



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

system were provided. This material is available free of charge via the Internet at http://pubs.acs.org.

REFERENCES (1) Famulok, M.; Mayer, G. Acc. Chem. Res. 2011, 44, 1349-1358. (2) Xiao, Z.; Farokhzad, O.C. ACS Nano. 2012, 6, 3670-3676. (3) Chen, J. H.; Zhang, J.; Li, J. Biosens. Bioelectron. 2010, 25, 996-1000. (4) Tan, W.; Donovan, M. J.; Jiang J. Chem. Rev. 2013, 113, 2842-2862. (5) Huang, Y.; Liu , X. Q.; Huang , H. K. Anal. Chem. 2015, 87, 8107-8114. (6) Chen, A. L.; Yan, M. M.; Yang, S. M. Trends Anal. Chem. 2016, 80, 581-593. (7) Liu, J. B.; Yang, X. H.; Wang, K. M. Anal. Chem. 2013, 85, 11121-11128. (8) McGown, L.B.; Joseph, M.J.; Pitne, J.B. Anal. Chem. 1995, 67, 663–668. (9) McKeague, M; DeROsa, M.C. Nucleic Acids. 2012, 748913. (10) Yang, C. N.; Spinelli, S; Perrier, E. Anal. Chem. 2015, 87, 3139–3143. (11) Wu C.C.; Yan L.; Wang, C.M. Biosens. Bioelectron. 2010, 25, 2232–2237. (12) Chen, J.H.; Fang, Z.Y. ; Lie, P.C. Anal. Chem. 2012, 84, 6321–6325. (13) Zuo, X. ; Xiao, Y.; Plaxco, K.W. J. Am. Chem. Soc. 2009,131, 6944–6945. (14) Bai, Y.F. ; Feng, F. ; Zhao, L. Analyst. 2014,139 ,1843–1846. (15) Jin, F. ; Lian, Y.; Li, J.S., et al. Anal. Chim. Acta. 2013, 799, 44–50. (16) Feng, L.Y.; Zhang, Z.J.; Ren, J.S. Biosens. Bioelectron. 2014, 62, 52–58. (17) Jiang, H.; Ling, K.; Tao, X., Biosens. Bioelectron. 2015, 70, 299–303. (18) Zhang, J.; Wang, L.H.; Pan, D. Small. 2008, 4, 1196–1200. (19) Wang, Q.; Huang, J.H.; Yang, X.H. Sensor Actuat B-Chem. 2011, 156, 893–898. (20) Park, J.H.; Byun, J.Y.; Shim, W.B. Biosens. Bioelectron.2015, 73, 26–31. (21) Xu, X.W.; Zhang, J.; Yang, F. Chem. Commun.2011, 47 ,9435–9437. (22) Li, M.; Yang, H.M.; Ma, C. Sensor Actuat B-Chem. 2014, 191, 377–383. (23) Kashefi-Kheyrabadi, L.; Mehrgardi, M.A. Biosens. Bioelectron. 2012, 37, 94–98. (24) Hu, K.; Huang, Y.; wang, S. E. J. Pharm. Pharmacol. 2014, 95, 164-168. (25) He, X.X.; Li, Z.X.; Jia, X.K. Talanta. 2013,111, 105–110. (26) Patei, D. J.; Curr. Opin. Chem. Biol. 1997, 1, 32-46. (27) Cui, H.; Zou, L.; Lin, N. Anal. Chem. 2012, 84, 5535-5541. (28) Zhang, D.; Lu, M.; Wang, H. J. Am. Chem. Soc. 2011, 133, 9188-9191. (29) Gokulrangan, G.; Unruh, JR.; Holub, D. F. Anal. Chem. 2005, 77, 1963- 1970. (30) Sandrine, P.; Corinne, R.; Valérie, G. Biosens. Bioelectron. 2010, 25, 1652–1657. (31)Wang, S. E.; Huang, Y.; Hu, K. Anal. Methods. 2014, 6, 62–66. (32) Zhu, Z. Y.; Schmidt, T.; Mahrous, M. Anal. Chim. Acta. 2011, 707, 191– 196. (33) Jiang, Y. X.& Tian, J. N. & Hu. K. Microchim Acta. 2014,181,1423–1430 (34) Zhu, Z. Y.; Ravelet, C.; Perrier, S. Anal. Chem. 2012, 84, 7203−7211. (35)Huang, Y.; Liu, X. Q.; Zhang, L. L. Biosens .Bioelectron. 2015, 63, 178–184. (36) Huang, Y.; Liu, X. Q.; Huang, H. K. Anal. Chem. 2015, 87, 8107−8114. (37) Xu, D.K.; Li, H.; Liu, X. ; Jia W. C. Lab Chip. 2016, DOI: 10.1039/C6 LC01208F. (38) Li, H.; Zhao, Y. J.; Chen, Z. Biosens. Bioelectron. 2017, 87, 428−432. (39) Li, H.; Hu, H. T.; Zhao, Y. J. Anal. Chem. 2015, 87, 3736−3745. (40) Li, H.; Hu, H. T.; Xu, D. K. Anal. Chem. 2015, 87, 3826−3833.


Page 10 of 16

Page 11 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(41) Li, H.; Zhu, Y.;Dong, S. Y. Anal. Chim. Acta. 2014, 829, 48-53. (42) Gabor, M. J Fluoresc. 2006, 16,511–524.

Scheme 1. Principle of the dual amplified aptasensor based on bivalent aptamers and Ag10NPs enhancement.

Table 1. Oligonucleotides Sequences used in this assay



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Two-dimensional structure of the intact aptamer Lac-A4 and split aptamers truncated into DNA-1 and DNA-2, DNA-3 and DNA-4, DNA-5 and DNA-6 respectively(a), FP value of different split aptamers with the concentration of 0 or 1 µg/mL Lac (b).


Page 12 of 16

Page 13 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Diagram of probes optimized process. Illustration(a) and FP value(b) of different paired bases with the concentration of 0 or 1 µg/mL Lac .

Figure 3. Verification of dual sites binding to Lac with the concentration of 0 or 1 µg/mL Lac, illustration(a) and FP value(b) .



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. FP/FP0 of different concentrations of probes with 5 µg/mL Lac (a); FP signal of Ag10NPs modified different length DNA chains with 0 or 1 µg/mL Lac (b).

Figure 5. FP signal of different numbers of modified DNA-2 chains per Ag10NPs by this assay with 1µg/mL Lac (a). Characterization of signal amplified ways with 0 or 1µg/mL Lac by this assay(b).

Figure 6. Specificity of the apatsensor, the concentration of negative proteins were 50 times as many as that of Lac (a); Linear relationship between Log C and ∆FP of the Lac-A4(b), capture probes and signal probes(c).

Table 2. Sample recoveries of low concentrations at different diluted ratio.


Page 14 of 16

Page 15 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Linear relationship of MeadJohnson enfinnitas (a), Beingmate Love (b), Carecoming Lactoferrin (c) samples by this assay. Actual detection of Lac-contained milk powered samples (d).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents (for TOC only)


Page 16 of 16

Bivalent Aptasensor Based on Silver-Enhanced Fluorescence

Recommend Documents