Suspension array platform based on aptamer for high-throughput detection of ve environmental hormones

As a common small molecule substance, environmental hormones widely exist in nature, especially water sources, which have a profound effects in humans. Highly ecient and sensitive method for estrogens in the environment are essential. In this paper, a novel high-throughput platform was established based on ve small hormones molecules specicity aptamer and magnetic beads (MBs). The results showed that the sensitivity of the proposed method are greatly improved. The limit of detection(LOD) of this method for atrazine(Atz), profenofos, bisphenolA(BPA), estradiol(E2), and polychlorinated biphenyls(PCBs) were 9.46, 20.75, 23.81, 8.97, 6.27 pg/mL, respectively. The Recovery rate of the diluted environmental hormones spiked in the samples of Haihe river were in the range of 87.5-111.02% with relative standard deviations (RSDs) lower than 28.44%. This platform based on new complementary strand fragments can simultaneously rapid detection ve environmental

the change of color, the disadvantage is that the quanti cation is not accurate enough. As a mature and common commercial immuno uorescence detection method, ELISA [14][15][16][17] could quantitative and qualitative detection environmental hormones. In recent years, various nanomaterials combination with ELISA and improved the sensitivity of method [18][19][20]. GC-MS is a very sensitive detection method which LOD can reach pg level. The disadvantage is instrument used in this method bulky and high requirement for operators. The simultaneous acquisition of multiple target signals in one sample is research hot topics, which is also absent from conventional detection methods. In recent years, aptamers combined with other technologies have been widely used in medical diagnosis [21,22], food [23], and environmental monitoring [24][25][26]. As a recognition molecule, aptamers can replace antibodies for microorganisms [27,28], cells [29], and heavy metal ions [30,31]detection. Compared with antibodies, aptamers have the advantages of higher modi ability, variable structures, easy ampli cation which suitable for highthroughput detection. More important, aptamers are rapidly synthesized [32], inexpensive and ideal alternatives to antibodies.
Single-stranded oligonucleotide sequences are optimized and screened through exponential enrichment (SELEX), and obtain the most suitable aptamers for detection [33]. Suspension array technology (SAT) differs from ordinary solid-phase chips. Two different dyes were used in different proportions to obtain 100 different colors MBs. The exible multiple-analyte system(xMAP®) can simultaneously detect 100 kinds of targets. The instrument can emit red light and green light at the same time, and the MBs can be analyzed quantitatively and qualitatively through these two kinds of light. At present, the suspension array technology is widely used in allergens [34], thrombin [35,36],etc. These aspects are mostly combined with nucleic acid ampli cation (PCR, RCA) methods to improve the sensitivity of detection.
In this work, ve environmental hormones were selected and coupled to the MBs of suspension array system for the rst time, and designed biotin modi ed complementary chain (biotin-cDNA) in the semi binding region (Fig. 1). Finally, the environmental hormones were qualitatively and quantitatively analyzed by identifying the mean uorescent intensity(MFI) of MBs through SAT platform. In our work, we innovatively designed biotin-cDNA in the half of the environmental hormones binding region, and the remaining design was in the adjacent region. The nal results showed super sensitivity. We nally completed the assay for detection ve environmental hormones in one hour. The application of aptamers, greatly reduced the cost of whole detection.

Chemicals and Reagents
Estradiol  (Table S1) and dilute to 10µM before use. Streptavidin phycoerythrin (SA-PE, 1 mg/mL) was supplied by Invitrogen

Speci c recognition
Five environmental hormones and their corresponding analogs were formulated into solutions with a concentration of 10 ng/mL. The 96-well plate was divided into ve groups, and each group was divided into targets and their corresponding analogs. Add 10 µL aptamer-coupled microspheres to the wells, then add 5 µL corresponding small molecules or the like, and incubate at 37°C for 0.5 hours. Then add the corresponding complementary strand and incubate for 0.5 hour in the same environment. After washing the plate, add SA-PE and react at 37°C for 0.5 hours, and nal results were read out by machine.

Recovery tests in river samples
To con rm the availability of the assay, we prepared ve small molecule standards solutions with a concentration of 1 and 10 ng/mL and add them to the Haihe river sample (the samples were collected in Haihe River, Tianjin, 117° 12′ 7′′ E, 39° 06′ 41′′ N). All the samples were ltered through 0.45 µm micro ltration membranes before test. Add 10 µL of 5 kinds of aptamer-coupled MBs to each well of the washed black opaque 96-well plate. Then add 5 µL of the spiked samples above. React at 37°C for 0.5 h, and then add 4 µL of biotin-cDNA to ensure that the nal concentration ratio of aptamer to biotin-cDNA is 1:1. Each sample was measured three times, and the test values were calculated based on the standard curves, we also calculated the recovery and relative standard deviations (RSDs) of each sample.

Results And Discussion
Experimental feasibility veri cation The ve aptamers came from SELEX screening results, and we also designed ve corresponding biotin-cDNA (all the sequences list in Table S1). A circular dichromatogram (CD) was used to verify the binding ability of small molecule standards and aptamers. It can be seen from the results that the small molecule standard has no obvious absorption peak, while the aptamer shows positive and negative peaks at 273nm and 245nm, respectively. When the aptamer is coupled with a small molecule standard, the peak amplitude will be signi cantly reduced, but the peak position will not change (Fig.S1). The combination of the aptamer and the small molecule standard will cause the conformational change of the aptamer, which leads to the change of CD amplitude.
The feasibility of this method is veri ed by uorescence experiment. Take estradiol as an example (Fig.2).
when E2 standard absence, the uorescence value is the highest. After adding different concentrations of targets, the uorescence value and the concentration of small molecule standards were negative correlated. This is because the concentration of the small molecule standard competes with the biotin-cDNA for the aptamer, and as the concentration of the standard increased, the biotin-cDNA bound to the aptamer decreases, resulting in a decrease of uorescence value. The result conformed to the experimental principle and proved the feasibility of the experiment.

Optimization of reaction conditions
During the reaction, we evaluated several parameters to achieve the best uorescence response. It can be seen from Fig.S2 that the difference of uorescence value is not signi cant under different MBs stock solutions, but in 0.1M PBS buffer, the uorescence value is the highest. Considering economy and universality, we chose 0.1M PBS as stock solution. As a costly reporter molecule, the concentration of SA-PE need to be considered. The results showed that when SA-PE was diluted 1:50 and 1:100, the MFI change not signi cant considering the cost, we nally determined the optimal concentration of SA-PE was 1:100 (Fig.3a). By optimizing these conditions, the measured results can reach the optimal value. We also explored the optimization of coupling conditions and the results showed that the optimal coupling time was 1 h (Fig.S2), and the optimal coupling temperature was 37℃ (Fig. 3b).
After con rming that the aptamer was successfully coupled to the MBs, the optimal coupling amount of the ve aptamers on the MBs was explored (Fig. 4). With the increase of aptamer concentration, MFI gradually increased and nally entered a smooth period or decreased. We select the point of maximum MFI as optimal coupling concentrations. The maximum coupling aptamer concentration for estradiol, atrazine, profenofos, polychlorinated biphenyls, and bisphenol A are 4, 2, 3, 5, 2μM respectively.

Characterization of surface modi cation of Aptamercoupled MBs
The MBs, MB-Apt, MB-Apt-cDNA complex were characterized by scanning electron microscope (SEM) and Zeta potential. The SEM can enlarge targets tens of thousand times and intuitively observe the structural changes on the surface of substance. The zeta potential informs the degree of bonding between chemical bonds through the change of potential [37]. We employ SEM to scan the surface of MBs (Fig. 5). From Fig. 5a we can see that the blank MB is rough surface, when coupled with aptamers, the surface of the MBs is covered with a thin layer of protrusions (Fig 5b). Fig. 5c showed the surface of the MBs which were wrapped by biotin-cDNA and small molecule standards. We can see irregularly shaped protrusions on the surface of MB, indicating the surface of MBs covered by the complex.
We also characterized the zeta potential to observe of the potential change of coupling MB. Take the example of E2 coupling MB (Fig. 5d), we can see that carboxylate blank MB exhibits a zeta potential of 90.7±0.52mV, the value signi cantly drops to 22.9±0.41mV after coupling with aptamer, because amino aptamer coupling with carboxyl group on the MB and neutralized the potential. When add biotin-cDNA and form MB-Apt-biotin-cDNA complex, the zeta potential is 24.9±0.73mV. The result indicated the combination of biotin-cDNA and aptamer did not cause obvious potential change.
The speci city of assay for the detection of environmental hormone Speci city is an important indicator to evaluate one assay. We selected two analogs for each of ve environmental hormones, and the concentration was 10 ng/mL. When ve targets were added to the detection system, the small molecules competed with the complementary chain for the corresponding aptamer, resulting in a decrease in MFI Fig. 6 . When the analog exists in the detection system, the analog cannot speci cally bind to the aptamer, and the uorescence value is not signi cantly reduced. It is proved that the ve environmental estrogens have good speci city and can be used in real samples detection. The stability and sensitivity of this method are veri ed. In addition, during the detection process, only need two-step washing. We can complete the assay in about 1.5 h.
The sensitivity of assay for the detection of environmental hormones After the conditions were optimized, different concentrations of small-molecule targets were added and the Logistic tting equation was used to establish a single-channel detection. It can be seen that all of the ve single-channel curves have an "S" shape. As the concentration of small molecule targets increased, the MFI gradually decreaseds, which is consistent with the experimental setup. By this method, the LOD of atrazine, profenofos, bisphenol A, estradiol, and PCBs were 8.5589, 16.8631, 23.2314, 8.4511, 6.2609 pg/mL (Table.1) respectively. The single-channel detection curve of these targets can be completed on a 96-well plate, and the detection time is between 40-45 minutes, which greatly shortened the detection time.
We obtained the optimal reaction conditions, and draw the multi-channel curve of ve environmental endocrines, intuitively obtained the linear regression curves of ve small molecule standards in SAT. The result showed a remarkable value, The LOD of Atz, Profenofos, BPA, E2, PCBs reached 9.465, 20.7514, 23.8094, 8.9712, 6.2677 pg/mL, respectively (Table  2). However, the simultaneous addition of ve small molecule standards in per well slightly affects the tness of standard curve (Fig.7), resulting in not all the determination correlation coe cient (R 2 ) being greater than 0.98. Table 1 showed the Equation, LOD and R 2 of ve small molecule standards.
We also compared this method with the classic ELISA experiment. The LOD of profenofos by ELISA was obtained experimentally, and the rest were cited in the literature. Through comparison, it can be seen that the detection limit of this method is lower than that of ELISA (Table 3), the LOD of this method was 20-400 times lower than that of ELISA.
Single-channel detection and multi-channel detection are the same for each type of small molecule coupled microspheres. Under the same coupling and detection conditions, the corresponding curves are obtained. Compared with single-channel detection, the number of probe microspheres contained in each well has been increased by ve times, and the content of other substances in each well is also as high as a dozen, making multi-channel detection theoretically more di cult. However, duo to the fact that SAT allow all the substances to be detected in the liquid phase, and the sampling is more uniform, the result present a high speci c result. Although the multi-channel detection system will be affected to some extent, it also forms a good linear relationship and a lower detection limit. Proved that the multi-channel detection system can be used for high-throughput and high-sensitivity detection. Spiked actual samples To investigate the ability of the method in practical application, we selected water from the Haihe River to carry out a spiked recovery test. The relative standard deviation of the actual sample detection is between 7.64-28.44%, and the relative standard deviation of estradiol is relatively high. In the case of highthroughput detection, all meet the detection standards (Table 4). It can be seen that this method can be used for the detection of estradiol, bisphenol A, polychlorinated biphenyls, atrazine, and profenofos in water. Of course, these ve targets can also be randomly matched for actual sample detection. The test can be performed on the premise of setting the method, and the detection of samples it only takes about 1 h to complete, and the number of MBs read in each sample well (usually 50 or 100) can be set. This detection is rapid and accurate, and has great practical application value. Conclusions Rapid detection techniques for food and water environmental contaminants are an hot topic of research. This is because toxins, endocrine disruptors, heavy metals present in nature will enter the human body through water sources, food, and trace concentrations will have a profound effect on the human body [39]. Despite the traditional monitoring methods playing a great role in daily detection, not fast enough [40], single-thoughput [41] are still the limiting factor that needs to be addressed. As a detection platform with mature applications in medicine, the suspension array technology has high sensitivity and speci city, which can be applied as a promising technology in the rapid detection of water environment and food products.
In short, we selected a new fragment complementary to the aptamer, and indirectly competed with the environmental hormones for the aptamer through the biotin-cDNA, and completed the high-throughput detection of ve targets. The selected new complementary fragments which modi ed with biotin improved the sensitivity of detection. It has been demonstrated that the unmodi ed aptamer reduced the cost of detection and increased the exibility of detection. Compared with the previous report [42], the sensitivity of ve small molecules is greatly improved and realized high-throughput detection.
In this study, we have demonstrated the great advantages of using aptamers instead of antibodies as probes for monitoring small molecules of environmental estrogen. A standard curve of the assay was constructed and analyzed. Small molecule commercial antibodies are expensive, hard to obtain, and the synthesis of short aptamer chains is convenient, quick, and inexpensive. The results also showed that the aptamer and small molecule had a good binding force, and the detection limit reached the pg level.
The SAT is green and simple. The assay can be performed in a two-step process utilizing a 96 well plate during the sample incubation stage and does not require special treatment by professionals. High throughput is undoubtedly a great advantage over conventional detection methods. The simultaneous detection of dozens of samples within 1.5h, including all the incubation, detection stages, greatly accelerates the progress and time.
In the eld of rapid detection, it is only of practical interest to be able to apply the method to on-site monitoring. The good or bad of water quality directly relates to human health. We selected Haihe Water, which is closely related to living, as a sample, and performed a spike recovery test, nally the satisfactory results were obtained. At different spiked concentrations, the nal recoveries rate were all between 87.5-111%, indicating that the established platform can be applied in real sample detection.
In summary, the combination of aptamers with SAT enables rapid, high-throughput, sensitive detection using minimal sample volumes, green and label free. This novel platform can be used as a tool to detect and establish small molecule solutions to environmental pollution. In addition, this system serves as a research tool for pollutant changes in the environment. Further technological development as well as advanced studies have shown that the system can be integrated into food and water environments for monitoring contaminant traces to prevent hazards to living organisms.

Declarations Supporting Information
We list the method used in our experimental. Figure S1 showed The CD chromatogram of ve environmental endocrine, Figure S2. The optimization of coupling conditions. Table S1 is  Schematic diagram of the indirect competition SAT for environmental hormones with complementary chains for high-throughput detection.

Figure 3
The optimization for (a) SA-PE dilution ratio, (b) Incubation temperature. The error bars represented the relative standard deviation of the three groups of paralleled measurements Figure 6 The Selectivity for possible interferences