Method to Determine the Optimal Aptamer-to-Bead Ratio by Using Flow Cytometry

Research on the effective attachment of aptamers to beads, which is essential for using aptamers, has made relatively little progress. Here, we demonstrate a new method based on flow cytometry to determine the optimal aptamer-to-bead ratio for aptamer immobilization. The fluorescence intensity increased with a gradual two-fold increase in the aptamer fluorescence concentration, peaked at an aptamer-to-bead ratio of 2.56 × 105, and tended to decrease at higher ratios. A similar pattern was observed in an additional analysis using fluorescence microscopy. However, measurement of the free aptamer concentration after the aptamer-bead conjugation reaction revealed a large aptamer loss compared to the 1.28 × 105 aptamer-bead ratio. In addition, the binding efficiency of the aptamer/bead to the target was highest at the aptamer-to-bead ratio of 1.28 × 105. Taken together, our data suggest that the proposed method is the best and easiest for determining the optimal aptamer-to-bead ratio.


Introduction
Flow cytometry is a popular cell analysis technique used in various felds. It was frst used in the 1950s to measure the volume of cells in a rapidly fowing fuid stream and was then developed to analyze cell characteristics [1]. Te technique is predominantly used to measure the fuorescence intensity produced by fuorescently labeled antibodies to detect target proteins. In addition, the technique has been used to recognize nonfuorescent cells by side and forward scatter through the detection of scattering at a 90°angle relative to the laser and along the path of the laser, respectively [2]. Te greatest advantage of fow cytometry is the high reliability of the results, which allows the rapid analysis of multiple samples and easy comparison with the control group [3]. In recent years, fow cytometry has been applied in various ways other than for cell analysis. For example, fow cytometry has been used to compare and measure the amounts of various proteins in cells [4]. It has also been used to quantify secreted proteins such as cytokines using beads with many diferent fuorescently labeled antibodies at the same time [5,6].
Tree independent groups of aptamers have been identifed. Te term "aptamer" refers to a short single-strand nucleotide (DNA or RNA) or peptide that can specifcally bind to the desired target [7][8][9]. Owing to the high specifcity of aptamers to their target molecules, aptamers have found applications in various felds such as biosensing, diagnostics, and therapeutic applications [10,11]. Various techniques for the systematic evolution of ligands by exponential enrichment (SELEX) have been used in many studies to discover aptamers that interact with specifc targets [12]. In the following study, aptamers discovered through SELEX should be attached to appropriate beads, either by passive adsorption or covalent coupling depending on the intended application [13]. Te beads are available in a variety of formats (polystyrene, agarose, and magnetic beads) and are coated with carboxyl groups, avidin, streptavidin, or other proteins. Magnetic beads coated with a linker arm for immobilization were frst used in 1997 [14]. Te coated beads can be used in various applications. For example, carboxylcoated polystyrene beads have been used for binding to amine-modifed aptamers and streptavidin-coated agarose beads have been used for binding to biotin-labeled antibodies [15]. Currently, aptamers are attached to beads either chemically or through a specifc interaction such as that between avidin and biotin.
However, comprehensive studies to determine the optimal aptamer-to-bead ratio have not yet been reported. Tis motivated our study, in which we used fow cytometry to determine the optimal amount of aptamer to form the aptamer-bead complex.
Te aforementioned complex is essential for aptamer studies. Some papers provide information about the reagents, but the molar ratio bead-aptamer is ill-defned [16]. Furthermore, most papers do not indicate the exact amount or percentage of aptamers and beads used [17]. Te optimal binding ratio of the aptamer is diferent for each experiment and depends on the binding method and size of the beads. Terefore, it is important to determine the optimal aptamerto-bead binding ratio in diferent experimental environments.  Table) with or without FAM (5′-Fluorescein phosphoramidite), selected as a specifc aptamer binding Ni 2+ /Co 2+ [18], were synthesized by Integrated DNA Technologies (IDT). Tis aptamer was selected because the average length of the metal ion-specifc aptamer is 80-100 m, and the available equipment could be used to confrm the amount of Ni 2+ /Co 2+ bound to the aptamer. Te amine group at the 5′-terminus was designed to allow covalent binding of the aptamer to the carboxyl groups on the CLB9 beads (Sigma), which were used to immobilize the aptamers. MES bufer (50 mM, pH 5.9) was used as the solvent in which to couple the aptamer to the beads and to subsequently wash the beads. For coupling, the carboxyl groups on the surface of the beads were activated using N-(3dimethylaminopropyl)-N-ethylcarbodiimide (EDC) (Sigma) and N-hydroxysulfosuccinimide (sulfo-NHS) (G-Biosciences). Cobalt (II) chloride was purchased from Sigma-Aldrich (St. Louis, MO, USA; 449776-5G).

Aptamer-Bead Complex.
To prepare the aptamer-bead complex, a 50-μL (2.5 × 10 11 , 0.9 μm mean bead size) aliquot of beads was transferred to a microtube and centrifuged for 4 min at 13,000×g. Te supernatant was carefully removed and discarded. Te bead pellet was resuspended in 200 μL MES bufer and vortexed thoroughly for 10 s. To activate the bead surface, 40 μL of 0.3 M EDC and 40 μL of sulfo-NHS 0.4 M solution were added, followed by mixing. After activation, diferent concentrations of the aptamer were added to the microtubes and the total volume was adjusted to 500 μL. Te coupling reaction was performed for 2 h in the dark at room temperature with rotation (Roto-Bot; Benchmark, USA). Te tubes were centrifuged for 4 min at 13,000×g. Te supernatant was used to measure the amount of free aptamer, and the pellet was resuspended in 500 μL PBS-T. Tis washing step was repeated thrice.

Free Aptamer Measurement.
Te fuorescence of the supernatants was measured using a Tristar 5 plate reader (Berthold, Germany). Absorbance data were collected and processed using the MikroWin2010 program (Labsis Laborsysteme GmbH). Te manufacturer's instructions were followed for operating the machine.

Flow Cytometry Analysis.
Beads with or without aptamer labeled with FAM were suspended in a total volume of 300 μL PBS, followed by analysis using the FACS Canto II fow cytometer (BD Biosciences, CA, USA). Data were collected using FACSDiva software (BD Bioscience, CA, USA) and subsequently analyzed using FlowJo software (FlowJo, OR, USA).

Fluorescence Image Capture.
Te fuorescently labeled aptamer-bead complex was analyzed using a CKX53 microscope (Olympus, Tokyo, Japan). After aptamer-bead coupling, a 5 μL aliquot of beads was transferred onto a slide glass and covered with the cover glass. Te microscope was controlled by a computer with the CellSens Entry software (Olympus) at 200× magnifcation. Photographic images were captured in a completely darkened room, and the entire process was completed within 10 minutes.
Te fuorescence intensity on the images was measured using ImageJ software (National Institutes of Health, MD, USA). First, Photoshop (Adobe Systems, CA, USA) was used to select regions of interest (ROIs), measuring 100 × 100 pixels from the images. ROIs of the same size were selected and cut with Photoshop in the same positions in all images to maintain a standard in the analysis of the measurements. Next, ImageJ software was used to extract the average brightness value from the ROI. Each pixel was assigned a numerical value that represented its brightness on the grayscale 22, ranging from 0 (black pixels) to 255 (white pixels).

ICP-OES Measurements.
After aptamer-bead coupling, 300 μL of 10 μM CoCl 2 was transferred to a microtube containing the aptamer-bead complex and the mixture was incubated for 10 min at room temperature with rotation (Roto-Bot) [19]. After incubation, the microtube was centrifuged for 4 min at 13,000×g, and the concentration of Co 2+ in the supernatant was measured by ICP-OES using a PlasmaQuant PQ 9000 spectrometer (Analytik Jena, Germany) equipped with a Standard Kit. Te data were collected and processed using Aspect PQ 1.2.4.0 software. Te manufacturer's instructions were followed for operating the machine.

Statistical Analysis.
All statistical analyses were conducted using SPSS software (IBM ® SPSS ® Statistics Ver. 23).
For multiple comparisons, the data were analyzed using oneway analysis of variance (ANOVA). Tukey's posthoc test was used to identify signifcant diferences among groups.

Flow Cytometry Can Detect Beads to Which Aptamers Are
Attached but Not Tose without Aptamers. Flow cytometry, in which the laser passes through the cells and is refected, uses pattern analysis to acquire information about the characteristics of cells. To explore whether fow cytometry could recognize polystyrene beads, we started our investigation by analyzing the beads without and with aptamers attached. Flow cytometry did not recognize the beads without aptamers; however, those with aptamers were detected (Figure 1(a)). Specifcally, polystyrene beads to which fuorescently labeled aptamers were attached, were clearly detected (Figure 1(b)). In general, the way fow cytometry perceives cells is to analyze three parameters depending on the degree of light scattering when the cell passes through the laser beam [3,20]. In other words, the front-facing light collecting forward scatter (FSC) represents the size of the cell, and the side scatter (SSC) collecting light from the side represents the intracellular granularity, and fnally, the fuorescence intensity [21]. Beads without an aptamer are analyzed by FSC, which entails detecting the laser light scattered from the front; however, because SSC, which is the lateral light used to measure the presence of granules in the cell, is not generated, these beads are not necessarily recognized by fow cytometry. However, in the case of an aptamer-coupled bead, fow cytometry would be able to recognize it because the light is difusely refected by the aptamer and collected by detecting the SSC.

Determination of the Aptamer-to-Bead Ratio by Flow
Cytometry. Because various types of beads are used in research with aptamers, it is inefcient to use the same aptamer-to-bead ratio for all beads. Terefore, determining the most efective ratio for each type of bead is essential. In our work, the aptamer-to-bead ratio was gradually increased from 5 × 102 to 5.12 × 105 by doubling the aptamer concentration incrementally while the concentration of beads was kept constant. As expected, the fuorescence intensity increased approximately 2-folds with a 2-fold increase in the aptamer concentration (Figures 2(a) and 2(b)). Tis analysis indicated that the aptamer-to-bead ratio of 2.56 × 105 was the most efective. However, interestingly, the fuorescence intensity tended to decrease at the higher ratio of 5.12 × 105 (Figures 2(a) and 2(b)). Te rate at which the fuorescence intensity increased became slower at the 6.4 × 104 ratio (Figure 2(c)). Tis result means that, above a specifc concentration of the aptamer (in this case, 6.4 × 104), the efciency whereby the aptamer binds to the bead is diminished. Tis may be due to the possible formation of nonspecifc conjugates between the larger number of aptamer molecules in a fxed volume of bufer. Taken together, these results indicate that the optimal aptamer-to-bead ratio is between 6.4 × 104 and 2.56 × 105.

Confrmation of the Aptamer-to-Bead Ratio by Fluorescence Microscopy.
To confrm the aptamer-to-bead ratio we determined by fow cytometry, we used fuorescence microscopy to analyze complexes containing fuorescently labeled aptamers and beads. Te fuorescence intensity gradually increased under the same conditions as those in Figure 2 (Figures 3(a) and 3(b)). An aptamer-to-bead ratio of 2.56 × 105 was found to be optimal, similar to the ratio determined using fow cytometry. We also confrmed that the fuorescence intensity decreased at higher ratios of 5.12 × 105, similar to the results obtained by fow cytometry (Figure 3(b)), and that the decrease was not excessive, similar to that observed by fow cytometry. Tis diference may be due to the fact that the samples measured using fow cytometry are fast fowing, which would result in all nonspecifcally bound aptamers becoming dislodged from the beads. Unlike fow cytometry analysis, however, the rate at which the fuorescence intensity increased started declining at an earlier point, i.e., at a ratio of 3.2 × 104 (Figure 3(c)). In the case of fuorescent images acquired using confocal microscopy, it is difcult to compare the exact intensity of light in diferent samples. Tis is because the overall brightness of the image is adjusted according to the intensity of the bright light. Terefore, the correction of the light intensity would have been applied from the ratio of 3.2 × 104 of which the image contains strong fuorescent spots (Figure 3(a)). Based on these data, the most efective aptamer-to-bead ratio was between 6.4 × 104 and 1.28 × 105, where the rate increase was more than twice.

Confrmation of Aptamer-to-Bead Ratio by Analysis of Free Aptamer Concentration.
Te results from the fow cytometry and fuorescence analysis suggested that it is not efective to continue increasing the aptamer-to-bead ratio beyond a certain value. To confrm this hypothesis, we analyzed the concentration of free aptamer that remained after exposure of the aptamer to the beads for complexation. Te concentration of the free aptamer gradually increased depending on the initial amount of aptamer (Figure 4(a)). At low aptamer-to-bead ratios (<6.4 × 104), the increase in the rate of the free aptamer is lower than twice, but at concentrations higher than a ratio of 1.28 × 105, the rate of Scientifca 3 increase is more than twice (Figure 4(b)). We also found that the rate increase at a ratio of 1.28 × 105 was the highest (Figure 4(b)), indicating that free aptamer molecules are more abundant after excessive aptamer-bead interaction. In addition, our data suggest that an excessive concentration of aptamers may result in aptamer molecules interacting with each other, which decreases the efciency with which they are attached to the beads.

Verifcation of Aptamer Activity at Each Aptamer-to-Bead Ratio via Inductively Coupled Plasma Optical Emission
Spectroscopy (ICP-OES) Analysis. Te data presented above indicate that binding of the aptamer to the beads was the most efective at a 1.28 × 105 aptamer-to-bead ratio. To verify this result, we determined the aptamer activity at each aptamer-to-bead ratio using ICP-OES analysis. For this test, we used an RNA aptamer that specifcally binds Co 2+ [18]  Scientifca and measured the concentration of Co 2+ remaining after Co 2+ binding to the aptamer-bead complex. Te rate at which Co 2+ was removed by the aptamer-bead complex was the highest at the aptamer-to-bead ratio of 1.28 × 105 (Figure 5), showing a pattern similar to our results described above. Although the aptamer-to-bead ratio of 2.56 × 105 was the highest, as determined by fow cytometry and fuorescence analyses (Figures 2 and 3), the aptamer-to-bead ratio was the most efective between 1.26 and 2.56 × 105 in experiments to remove real targets (cobalt), as shown in Figure 5. We also conducted experiments with various aptamers of similar lengths to see if the optimal ratio derived from this experiment could be applied to other aptamers (Supplementary Table). Te experiments confrmed that the results of the paper are applicable to similar aptamers with the same size beads (Supplementary Figure). Tese results indicate that the use of fow cytometry and fuorescence analysis to determine the aptamer-to-bead ratio is sufciently accurate. Several factors, such as the bead size, aptamer length, and bufer conditions, need to be considered when research involving the use of an aptamer is frst started. Te method proposed in this study is therefore useful in that  it enables the optimal aptamer-to-bead ratio to be determined under various initial conditions.

Conclusion
In conclusion, we developed a new method that relies on fow cytometry to determine the efective coupling ratio between aptamers and beads. Although it is important to have as many aptamers attached to the beads as possible, the efectiveness of the aptamer-bead complexes is more important. In this respect, this study determined the most efective aptamer-to-bead ratio using fow cytometry and various other methods. Flow cytometry analysis revealed that an aptamer-to-bead ratio of 2.56 × 105 enables the largest amount of aptamer to bind to the beads. We also found that an aptamer-to-bead ratio higher than 2.56 × 105 signifcantly reduced the amount of aptamer attached to the beads. Tis fnding was corroborated using other fuorescence techniques. In addition, our measurements of the activity of bound aptamers confrmed that an excessively large ratio of aptamer to beads (e.g., 5.12 × 105), has the efect of decreasing the binding efciency between the aptamer and the target. Tis experimental method is expected to be useful for determining the combination of various aptamers and beads in the future.

Data Availability
Te data used to support the fndings of this study are within the paper.

Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Supplementary Materials
Supplementary   Figure 5: Verifcation of aptamer Co 2+ binding capacity at each aptamer-to-bead ratio by ICP/OES analysis. Te concentration of Co 2+ ions was measured by ICP/OES after exposure to the indicated aptamer-bead complex. All experiments were repeated three times. Te removal rate is expressed as a percentage (passing through/initial ion concentration). Error bars represent standard deviations of replicates (n � 3). * * * Statistically signifcant diference in removal rate compared to control (P value <0.001).