Multifunctional Screening Platform for the Highly Efficient Discovery of

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Multifunctional Screening Platform for the Highly Efficient Discovery of Aptamers with High Affinity and Specificity Shao-Li Hong, Ya-Tao Wan, Man Tang, Dai-Wen Pang, and Zhi-Ling Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017

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Analytical Chemistry

Multifunctional Screening Platform for the Highly Efficient Discovery of Aptamers with High Affinity and Specificity Shao-Li Hong, Ya-Tao Wan, Man Tang, Dai-Wen Pang, Zhi-Ling Zhang*,† †

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of

Education), College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, People’s Republic of China.

ACS Paragon Plus Environment

Analytical Chemistry 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

ABSTRACT: Aptamers have attracted much attention as next generation of affinity reagents. Unfortunately, the selection efficiency remains a critical bottleneck for the widespread application of aptamers. Herein, to accelerate aptamers discovery, a multifunctional microfluidic selection platform was developed, on which the selection efficiency was greatly improved and high affinity and specificity aptamers were generated within two round selections. The multifunctional screening platform, precisely manipulating magnetic beads on micrometer scale, improved selection performance based on microfluidic continuous flow and enhanced the selection process control via in-situ monitoring and real-time evaluation. This method could suppress ~50-fold nonspecific binding nucleic acids compared to the conventional methods, further eliminate weakly bound nucleic acids within 9 min, simultaneously perform the negative selection and positive selection. And the selection effectiveness was in-situ and real-time monitoring. Three aptamers showed high affinity and specificity toward Mucin 1 (MUC1) with dissociation constants (Kd) in nanomolar range (from 22 to 65 nM). Furthermore, the selected aptamer was able to specially label cancer cells and efficiently capture exosomes with 64% capture efficiency. It demonstrated that the multifunctional screening platform was an efficient method to generate high-quality aptamers in a rapid and economic manner.


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Affinity reagents that specifically bind to their target molecules are a critical tool for biological and medical research. Nucleic acid-based aptamers exhibit significant advantages in this domain, because they are small size, synthetic accessibility and easy chemical modification.

1-2

Nowadays, they have already provided new

opportunities for the study of proteome,

3

pathogen

4

promising agent for cancer diagnostics and therapeutics.

and cell, 6-7

5

and are also a

Moreover, the discovery

of aptamers is performed in vitro rather than relying on in vivo biological processes, making them potentially well suited for high-throughput screening.

8

However,

conventional aptamer discovery by means of systematic evolution of ligands by exponential enrichment (SELEX) is time-consuming and labor-intensive. 9 Thus, it is still a challenge for developing a selection method that can rapidly generate high-quality aptamers with minimal resources. To accelerate aptamers discovery, a wide variety of molecular separation techniques have devoted to enhance the separation efficiency, including traditional filter-binding assays,

10

affinity chromatography,

microfluidic technology.

15-18

11

flow cytometry,

12

magnetic beads

13-14

and

Among of them, microfluidic technology offers many

unique advantages for molecular separation over conventional analytical techniques such as reduced sample and reagent consumption, high throughout, portability and potential for automating sample preparation and analysis.

19

Therefore, the aptamers

of selection in the microfluidic platform have attracted increasing attention. More recently, many microfluidic techniques have been explored to increase its separation efficiency, including capillary electrophoresis (CE) microfluidic SELEX,


20-21

sol-gel


microfluidic SELEX,

22

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beads-based microfluidic SELEX,

beads-based microfluidic SELEX.

24-25

23

and magnetic

In particular, magnetic beads-based

microfluidic SELEX has showed remarkable separation efficiency.

26-29

Nonetheless,

it is reported that the magnetic beads-based microfluidic SELEX is still lack of methods for in-situ monitoring and real-time evaluation the enrichment of aptamer. 30 In-situ monitoring and real-time evaluation could enhance the control of selection process to short the selection time. More importantly, it can avoid the selection blindness and reduce failure selection trials. Therefore, it is most desirable to develop a screening platform that possesses both high separation efficiency and in-situ monitoring and real-time evaluation ability. In our group’s previous works, we have developed some methods to generate the magnetic nanospheres (MNs) patterns on the micrometer scale. The MNs patterns chips could undergo continuous stringency washing, and had been applied to on-line detection of the cancer biomarkers and pathogens.

31-32

Additionally, the MNs were

fabricated by LBL (layer-by-layer) method according to our group’s work, which showed low nonspecific adsorption, fast binding kinetics and high magnetic response. 33-34

Thus, we took the advantage of MNs patterns chips to establish a multifunctional

screening platform. Aided by the screening platform, notable selection efficiency was achieved through the following factors. First, the selection incubating process was conducted in a flowing stream to suppress nonspecific binding nucleic acids, exhibiting ~50-fold decrease in DNA binding yield than conventional incubating method. Second, the rigorous washing effectively eliminated weakly bound nucleic


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acids within 9 min. Third, the platform simultaneously performed the negative selection and positive selection to improve the selection specificity. In addition, in-situ monitoring and real-time evaluating the selection process enhanced the control of selection process and avoided the selection blindness. As a model to demonstrate the superiority of the method, we selected MUC1 as a target protein. The MUC1 protein is a well-characterized large transmembrane glycoprotein that may potentially serve as the target for anticancer therapy.

35

The high affinity and specificity aptamers

against MUC1 were achieved only by two rounds selection, and the selected aptamer could be used to specially image cancer cell and further applied in exosomes capture with 64% capture efficiency. EXPERIMENTAL SECTION Fabrication of the Microfluidic Chips. The screening platform was consisted of selection chip and quantitative evaluation chip. The selection chip was fabricated according to our previous work.

32, 36

Supplemental Note S1 provides information on

chemicals and instruments used. Firstly, a square-profile channel mould was fabricated using AZ50XT photoresist to make the microvalves channel. Subsequently, PDMS components of part A and B were mixed at a ratio of 10:1 and poured onto the silicon master. After baking at 75°C for 4 h, the cured PDMS was peeled off the mould and punched inlets. Secondly, the fluidic channel was fabricated using the same soft lithography. Then PDMS components were spin-coated on the photoresist mould at a speed of 1200 rpm/min to fabricate the fluidic channel. Next, the valve control layer and fluidic layer were bound together. The two layers were baked at



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75 °C for 30 min, and then peeled off the photoresist mould to punch the inlets and outlets. Lastly, the nickel patterns on the indium tin oxide (ITO) glass were fabricated by the lithography and electroplating method. Briefly, AZ9260 photoresist was used to fabricate the mould, followed by an electroplating process to fabricate the nickel patterns. After the lift-off process, the nickel patterns were encapsulated in a layer of thin PDMS. Finally, the valve control layer, the fluidic layer and the PDMS-encapsulated nickel patterns layer were bound together to obtain the integrated selection chip. For the quantitative evaluation chip, it was fabricated according to our previous work.

31

In brief, the fluidic channel was fabricated using a 40-µm-thick positive

AZ50XT photoresist. Then two handmade miniature bars guiding magnetism were placed on the position beside the central channel with an angle of 120° and was perpendicular to the microfluidic channel. PDMS components were poured onto the AZ50XT master pattern. After baking at 75 °C for 4 h, the cured PDMS layer was peeled off the master, and punched the inlets and outlets. Finally, the cured PDMS layer and a microscope cover glass were irreversible bonded after being treated by oxygen plasma. Selection of the Aptamer. The whole of SELEX process began with the random DNA library. The HPLC-purified initial DNA library（~200 pmol）contained a central randomized sequence of 40 nucleotides (nt) flanked by 23 nt primer hybridization sites,

and

FAM

(6-carboxyfluorescei)

modified

the

forward

primer

(5’-FAM-AACCGCCCAAATCCCTAAGAGTC(N40)CACAGACACACTACACAC


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GCACA-3’). The DNA library in binding buffer [(20 mM Hepes, 150 mM NaCl, 2mM KCl, 2 mM MgCl2, 2 mM CaCl2 (pH 7.4)] was heated at 95 °C for 10 min, then quickly cooled down to 0 °C on ice for 10 min, and subsequently incubated for 5 min at room temperature. The negative protein (BSA) and positive protein (MUC1) were captured in the negative selection unit and positive selection unit on the selection chip, respectively. Then, the prepared library was injected into the chip by the velocity of 2 µL/min for 2 h. After incubating, washing buffer (1×binding buffer, 0.05% Tween 20) was used to wash the microchannel at a flow rate of 2 µL/min. Meanwhile, the selection process was monitored under an inverted fluorescence microscope. Finally, the aptamers-bound MNs were collected and amplified through PCR to use for the next round of selection. For the PCR amplification, we prepared a PCR mixture containing 36 µL of nucleic acid free water, 2 µL of 10 µM FAM labeled forward primer, 2 µL of 10 µM biotinylated reverse primer, 1 µL of taq DNA polymerase, 4 µL of dNTPs and 5 µL of DNA sample collected from the chip. The thermal cycling conditions of PCR were as follows: 95 °C for 10 min (initial denaturation), 30 cycles of 95 °C for 30 s, 65 °C for 30 s, 72 °C for 40 s, and terminated by an extra extension at 72 °C for 10 min. The optimal PCR amplification cycle number was determined by resolving PCR products on 3% agarose gel. When the procedure of PCR was finished, 5 µL of PCR mixture was removed and resolved on 3% agarose gel to monitor successful amplification and the correct size of the DNA fragment. After amplification, the biotin-labeled double-stranded PCR product was incubated



with streptavidin coated MNs at room temperature for 1 h to separate the dsDNA from the PCR mixture. After denaturing with 0.1 M NaOH for 20 min, the FAM-conjugated sense ssDNA strands were separated from the biotinylated antisense ssDNA strands by streptavidin-coated MNs, and purified by illustra Microspin G-25 Columns to use for the next round of selection or quantitatively evaluating the enrichment. Binding Affinity Evaluation for Selected Products of Each Cycle. To evaluate the enrichment of aptamers, 50 pmol ssDNA of each round was flowed into the quantitative evaluation chip at the rate of 0.2 µL/min for 2 h, where the target protein coated MNs were captured in the chip. After the incubation, washing buffer was pumped into the channel at the rate of 0.2 µL/min, and the fluorescence images were acquired using a Nikon inverted fluorescence microscope equipped with a high-sensitivity digital camera. The fluorescence intensity was analyzed by IPP software. Cloning and Sequencing of Selected Aptamers. After two rounds of selection, PCR products were amplified with unlabeled primers and purified using the DNA gel extraction kit. The purified samples were sent to Sangon Biotechnology Co., Ltd. (Shanghai, China) for cloning. Fifty colonies were randomly picked and sequenced at the Sangon. The sequences were analyzed using the software DNAMAN 6.0. The secondary structure analysis of selected aptamers was performed with the Internet tool Mfold software (http://frontend.Bioinfo.Rpi.edu/applications/mfold/). Affinity and Specificity Measurements. The average dissociation constant of individual ssDNA aptamers was measured via a fluorescence binding assay. The


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FAM-labeled ssDNA pools were diluted to several different concentrations (from 0 to 150 nM) in 30 µL of binding buffer. These dilutions were heated to 95 °C for 10 min and rapidly cooled on ice for 10 min, and then incubated for 10 min at room temperature. Subsequently, these various heat-treated ssDNA solutions were inhaled into the quantitative evaluation chip at a rate of 0.2 µL/min for 2 h and incubated with protein coated MNs. After this incubating, the mean fluorescence intensity of the MUC1-aptamer complex was used to evaluate the binding affinity by subtracting the mean fluorescence intensity of the unselected initial library. Using SigmaPlot software 12 (Jandel Scientific), the Kd of the aptamer−MUC1 interaction was obtained by fitting the dependence of fluorescence intensity verse the concentration of aptamer to the one-site saturation equation (1). 37 Y = Bmax X/ (Kd +X)

(1)

Where Y is the fluorescence intensity, X is the concentration of the ssDNA and Bmax is the fluorescence value at saturation. The specificity of the sequences was then analyzed by comparing their ability to distinguish between MUC1 and other proteins [such as BSA, human serum albumin (HSA), carcinoma embryonic antigen (CEA), a-fetoprotein (AFP) and prostate specific antigen (PSA)]. These proteins coated MNs were immobilized in the quantitative evaluation chip, respectively. Then aptamer was incubated with these proteins at a flow of 0.2 µL/min for 2 h, and the fluorescence intensity of bound ssDNA was determined by the fluorescence measurement. Cells Labelling and Exosomes Capture with the Selected Aptamers. The selected



aptamer was used for cells labeling experiments. Supplemental Note S2 provided information on breast cancer (MCF-7), human lung carcinoma (A549), T cells and human skin keratinocytes (HaCaT) cells culture. After incubating with 200 nM FAM-labeled aptamer for 1 h in the dark, the supernatant were removed and washed three times by the binding buffer. And then, the fluorescence images of cells were acquired by laser confocal fluorescence microscopy (Andor Revolution XD). On the other hand, the selected aptamer was used for exosomes capture experiments. Exosomes was collected by ultra-centrifugation from the cancer cell MCF-7. The amino group modified aptamer was synthesized from Sangon Biotechnology, and then the aptamer-coated MNs were incubated with exosomes. After magnetic separation, the complex was washed three times by the binding buffer and stained with the membrane

probe

DiI

(1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyaine

perchlorate). Fluorescence images of exosomes were acquired by laser confocal fluorescence microscopy. Capturing efficiency was calculated by the protein quantification. RESULTS AND DISCUSSION Design of the Multifunctional Screening Platform. The multifunctional screening platform was consisted of two parts: selection chip and quantitative evaluation chip. As shown in Figure 1A, selection chip was composed of fluidic channel (Red) and microvalve channels (Blue), which made the selection process integration. Fluidic channel was divided into two selection units including negative selection unit and positive selection unit, where nickel patterns were located below the fluidic channel


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and generated a high magnetic field gradient via magnets. MNs could be captured between the nickel patterns in the fluidic channel, and the captured MNs were arranged in a cross patterns corresponding to the nickel patterns (Figure 1B). The characterization of MNs was demonstrated in Figure S1. Microvalve channels were used to automatically control selection process by controlling the valves on-off. The detailed information about microvalve controlling procedures was illustrated in Figure S2. On the other hand, quantitative evaluation chip was mainly used to evaluate the selection effectiveness. As shown in Figure 1A, quantitative evaluation chip was comprised of single microchannel and two handmade miniature bars guiding magnetism, at which MNs were captured between the middle of microchannel. It should be noted that the quantitative evaluation chip can be high-resolution observation by the oil immersion lens (Figure 1D), leading to less sample consumption (1 µL MNs) and high signal acquisition ability.

Figure 1. (A) Photograph of selection chip and quantitative evaluation chip. (B) Microscopic image of MNs captured by selection chip. (C) Microscopic image of MNs captured by quantitative evaluation chip.



Overview of Multifunctional Screening Platform. The principle of multifunctional screening platform was shown in scheme 1. Briefly, bovine serum albumin (BSA) coated MNs and MUC1 coated MNs were separately injected into the selection chip and captured in the negative selection unit and positive selection unit, where BSA was applied as negative protein and MUC1 was used as positive protein. Next, an initial library of ssDNA was pumped into the selection chip for negative selection, at which a part of ssDNA was bound to BSA protein to reduce unspecific binding nucleic acids toward MUC1 (Scheme 1, step 1). And then the unbound library flowed through the positive selection unit for positive selection, where aptamer candidates were bound to target protein MUC1 and nonbinders were discarded (step 2). Meanwhile, the selection process was in-situ monitored under an inverted fluorescence microscope, and fluorescence signal was observed before and after incubating with library to guide the selection process (step 3). After incubating the library with the target protein, washing buffer was used for high rigorous washing the microchannel, where the weakly bound nucleic acids were eliminated (step 4). After that, the target protein-bound aptamers on MNs were collected and directly amplified by PCR with FAM- or biotin-labeled primers to use for the next round selection or sequence (step 5). Subsequently, 100 mM of NaOH was used to denature dsDNA into ssDNA for generating an evolved ssDNA library, and the evolved ssDNA library consisted of the FAM-conjugated sense ssDNA strand was separated from the biotinylated antisense DNA strand by streptavidin-coated MNs (step 6). Next, one part of the evolved ssDNA library was inhaled into the quantitative evaluation chip to evaluate the


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enrichment of each round selection through the fluorescence intensity, where the aptamer candidates of evolved ssDNA library could specifically bind MUC1 coated MNs in the microchannel (step 7). If the fluorescence intensity of enrichment was no obvious increased, the selection was stopped and PCR products were cloned and sequenced (step 8). The sequences were chosen for further analysis and application.

Scheme 1. Workflow of the multifunctional screening platform.

Improving

Selection

Efficiency

through

Microfluidic

Continuous

Flow

Controlling. To apply high-stringency selection condition, the random ssDNA library was incubated with the negative protein and positive protein in a flowing stream, which offers higher selection pressure over conventional stationary incubating method using an Eppendorf Tube (EP) under shaking condition. To quantitatively compare the efficiencies of different incubating strategies, we used real-time PCR (RT-PCR) to separately measure the amount of remaining DNA after incubation in a flowing stream and Eppendorf Tube (Figure 2A). And then we used the comparative CT



method (difference in threshold cycle value between the sample in a flowing stream and the sample of Eppendorf Tube) to quantify difference in template copy number of samples obtained from the two method.

38

The result showed the amount of nucleic

acids in a flowing stream decreased ~50-fold than nucleic acids samples in a stationary incubating condition (for example, Eppendorf Tube). This phenomenon suggested that the selection condition in a continuous flow suppressed non-specific nucleic acids binding against the target protein. After incubating the library with the target protein, washing buffer was used for high rigorous washing to further eliminate the weakly bound nucleic acids. Rigorous washing removed weakly or nonspecifically bound nucleic acids to select high affinity aptamers.

26

Compared with the conventional washing method by separation

technologies, the MNs patterns chip was more adaptable to stringently wash via the continuous microfluidic flow. During the process of washing, we in-situ monitored fluorescence intensity change with the washing time. The result showed that fluorescence intensity decreased from rapidly to slightly within the 9 min (Figure 2B), and it could be further supported by the polyacrylamide gel electrophoresis (PAGE) analysis (Figure S3). As a control, the fluorescence intensity indicated no obvious change when it didn’t apply continuous washing (Figure S4). We thus believed that the continuous flow washing imposed on the microchannel was an effective strategy to eliminate the weakly bound nucleic acids.


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Figure 2. (A) Difference in threshold cycle value between the sample and negative control with no template DNA in different incubating methods. (B) Histogram of fluorescence intensity with different washing time. Error bars correspond to standard deviation (n = 3).

After rigorous washing, target-bound and unbound nucleic acids were separated. As the separation of target-bound and unbound nucleic acids is the crucial step for successful aptamers selection, we characterized the partition efficiency (PE) of the selection chip after the high rigorous washing, which is widely used as a benchmark for molecular separation methods.

39

In this selection chip, we define PE as the ratio

between the amount of DNA collected through the outlet and those binding on MNs through quantitative PCR. The PE value of this method was (1.0±0.3) × 105 (Figure S5), which was exceeded about 100 times than those of the best conventional methods



(e.g., filters and columns).

21

In summary, this high-purity molecular separation was

enabled by continuous flow selection condition and high rigorous washing. Enhancing of the Selection Efficiency by In-situ Monitoring and Real-time Evaluation., The stable MNs pattern enabled us to in-situ monitor the selection process and real-time evaluate the enrichment effectiveness in the screening platform. In-situ fluorescence signal was used to monitor the whole selection process. As shown in Figure 3A, there was no fluorescence signal before the library injecting into the microchannel, and then fluorescence signal was gradually increased with the library flowing through the target protein. Finally, the obvious fluorescence signal was observed. The in-situ monitoring results were further verified by conventional PCR method. According to the gel electrophoretogram PCR analysis results (Figure 3B), no target band was observed before selection (Lane 3), while target fragment was found after selection (Lane 2). Importantly, it avoided selection blindness and failure selection trials, which was waste of time and chemicals. On the other hand, the enrichment effectiveness could be real-time evaluation. As shown in Figure 3C and D, the fluorescence intensity of target-specific aptamers was significantly increased with the increase round in the SELEX. Especially, the signal significantly changed after the first round selection, which indicated that the method had a rapid enrichment of aptamers. Compared with conventional evaluation methods by fluorescence spectrophotometer or surface plasmon resonance (SPR) instrument which required complex operation and long-time monitoring, real-time quantitative evaluation shorted the selection time and simplified the operation processes.


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Figure 3. (A) Fluorescence images of selection process. (a) before selection, (b) in the middle of selection, (c) after selection. (B) Gel electrophoretogram results. Lane 1: 50 bp DNA marker; Lane 2: the PCR product of MNs-bound aptamer after selection, Lane 3: the PCR product of MNs before selection. (C) Fluorescence images of each round selection. (D) Histogram of fluorescence intensity of each round selection. Error bars correspond to standard deviation (n = 3).

Improving Selection Specificity through Negative Selection. In order to improve the aptamer specificity, the negative selection unit was integrated in the selection platform. As shown in Figure 4, the negative selection unit bound much nucleic acids, thus a large number of unspecific nucleic acids was removed before binding the target protein MUC1. Meanwhile, the positive selection unit also showed obvious fluorescence signal. This indicated that the selection platform could simultaneously perform the negative selection and positive selection. It is difficult to efficiently screen aptamers with high affinity and specificity.

40

The integration of negative

selection on the platform offered an accessible method to isolate the aptamers with high affinity and specificity.



Figure 4. Microscopic images of negative selection unit (A) and positive selection unit (B).

The Multifunction Integration Based-on High-Precision Controlling of the Magnetic Field Distribution on the Micrometer Scale. The multifunctional screening platform, integrating microfluidic continuous flow, in-situ monitoring, real-time evaluation and negative selection, depended on high-precision magnetic field controlling on the micrometer scale. Therefore, it was necessary to map the magnetic field and force distribution on the micrometer scale. Finite element numerical simulations assisted by the software of COMSOL multiphysics were carried out to simulate the magnetic field and force distribution on the micrometer scale, and the detailed simulation and calculation process was described in Supporting S7. The simulated results showed that the nickel patterns generated high magnetic flux density (B) and magnetic field gradient (B·▽) (Figure 5A). The calculations of |(B·▽)B| demonstrated that the short-range gradient had a magnitude of 4 × 104 T/m within 50 µm in the platform (Figure 5B). It demonstrated the magnetic force (Fmag) was on the order of nano-Newtons, which was sufficient to tolerate the fluidic drag under the continuous flow condition. As a consequence, the multifunctional selection conditions were able to be completed in the selection platform. Meanwhile, random


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ssDNA library was flowed into the microchannel to generate solution mixing itself due to cross MNs patterns. As shown in Figure 5C and D, the simulation results showed the fluid flow had a strong vorticity magnitude when the library was injected into channel at a flow rate of 2 µL/min. Small-scale mixing is of uttermost importance in bio- and chemical analyses using microfluidic chip,

41

mainly because the liquid

mixture could improve the reaction efficiency.

Figure 5. (A) Numerical simulation of the magnetic field distribution with nickel patterns. (B) Calculations result of |(B·▽) B| vs the distance along the Y-axis direction. (C) Numerical simulation of the vorticity magnitude distribution in the selection chip. (D) Calculations result of the vorticity magnitude vs distance along the X-axis in the selection chip.

High Affinity and Specificity with Aptamer toward MUC1 through the Selection Platform. From the above real-time evaluation results, the enrichment signal reached a plateau in the next two rounds. Thus fifty colonies were randomly picked out and sequenced after two rounds selection. The sequences were analyzed by using the software DNAMAN 6.0 and distributed into three families based on their homology.



Three sequences (T1-6, T1-20, T1-36) from different families were chosen (Table S1) and determined their affinity. As shown in Figure 6A, the Kd values of T1-6, T1-20 and T1-36 were 44±13.6 nM, 22±7.2 nM and 65±15.0 nM, respectively, and the results were further confirmed by fluorescence spectroscopy (Figure S6). And then the selectivity of T1-20 aptamer with the lowest Kd was analyzed by comparing its ability to distinguish between MUC1 and other proteins (such as BSA, HAS, CEA, PSA, AFP). As shown in Figure 6B, the T1-20 was specifically bound with MUC1 and showed minimal binding to other five proteins. The secondary structure analysis was performed by Mfold software (Figure 6C). Interestingly, we found the central sequence of 40 nucleotides kept exactly the same secondary structure before and after truncating flanking primer sites (Figure S7). This could be presumably explained that the aptamer had a stability secondary structure. In a word, the selected aptamers with high affinity and specificity was directly supportive of the high efficiency of multifunctional screening platform.

Figure 6. (A) Determination of the dissociation constant T1-6, T1-20 and T1-36. (B)


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Determination of specificity for T1-20 aptamer. (C) Secondary structure analysis of T1-20 aptamer using Mfold software. Error bars correspond to standard deviation (n = 3).

Cells Labelling and Exosomes Capture with the Selected Aptamers. To further demonstrate the selected aptamer with an application potential, the T1-20 aptamer was used to label cancer cells and capture the cancer cell derived exosome. As shown in Figure 7 (A-D), the MUC1-positive cell lines of MCF-7 breast cancer and A549 lung cancer cells displayed high fluorescence signals after incubating with the FAM-labeled T1-20 aptamer. As a control, the MUC1-negative cell lines of T cells and human skin keratinocytes (HaCaT) cells showed no obvious fluorescence signal. The result confirmed that the T1-20 aptamer could selectively differentiate MUC1-positive cell lines from the negative cells. On the other hand, the T1-20 aptamer was used to capture the exosomes expressed with MUC1. The exosomes can be a new delivery system for tumor antigens in cancer immunotherapy.

42

In order to

immobilize the aptamer on the MNs surface, the T1-20 sequence was modified with the amino group so that it was used to conjugate with the carboxylic acid groups of MNs via carbodiimide chemistry. And then aptamer-coated MNs were used to capture the exosomes, while non-aptamer MNs were used as the negative control. After the capturing, membrane probe DiI was used to stain with the complex. As shown in Figure 7E and 7F, the red fluorescence on aptamer-coated MNs could be seen, while no obvious fluorescence in the negative control. The result clearly showed that the aptamer could be efficiently used to capture the exosomes which expressed the MUC1. And the capture efficiency was further calculated up to 64±1.2% by the protein



quantification (Figure S8). These results indicated that the selected aptamer could be used to establish an aptamer-based method to image cancer cells and capture exosomes, which would facilitate the development of cancer cell imaging, and novel cancer therapy.

Figure 7. (A-D) Confocal microscopic images of MCF-7, A549, T and HaCaT cells labeled with FAM-modified aptamer. (E-F) Confocal microscopic images after exosomes captured by aptamer-coated MNs or non-aptamer MNs and stained with DiI (excitation 549 nm, emission 565 nm).

CONCLUSIONS In summary, we have developed a multifunctional screening platform based on precisely manipulating MNs on micrometer scale. The platform performed the positive selection and negative selection simultaneously, conducted the selection in a


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flowing stream to suppress much more nonspecific binding than conventional incubating methods, and further imposed high-stringency selection condition by continuous washing to eliminate weakly bound nucleic acids. Meanwhile, in-situ monitoring the selection process could avoid the selection blindness, and the selection effectiveness was real-time quantitative evaluation. These efforts improved the selection performance and made the procedure of selection become controllable. Using the multifunctional screening platform to accelerate aptamers discovery, the high affinity and specificity aptamers of MUC1 were achieved only by two rounds selection, compared with previous reports that had a four rounds or ten rounds selection for MUC1 aptamers. 43-44 Furthermore, the selected aptamer could be used to specially image of cancer cell, and further applied in exosomes capture with 64% capture efficiency. In this way, we believe the selection platform offers a highly accessible method to generate high-quality aptamers.



ASSOCIATED CONTENT Supporting Information Figure S1, characterization of MNs and MNs-MUC1. Figure S2, valve-controlled process. Figure S3, PAGE analysis. Figure S4, fluorescence intensity with different time. Figure S5, standard curve of the threshold values. Figure S6, determination of the dissociation constant by fluorescence spectrophotometer. Table S1, sequences of significant homology. Figure S7, secondary structure analysis. Figure S8, BSA standard curve of different protein concentrations. Corresponding Author Email: [email protected]. Tel: 0086-27-68756759. Fax: 0086-27-68754067.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (21475099), the National Basic Research Program of China (973 Program, 2013CB933904), and the Natural Science Foundation of Hubei Province (2014CFA003). We are grateful to Lian Zhu, Di Dong and Xu-Yan Ma for their support in cell experiments. REFERENCES

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Table of Contents (TOC) Graphic


Multifunctional Screening Platform for the Highly Efficient Discovery of

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