Preparation and evaluation of highly hydrophilic aptamer-based hybrid affinity monolith for on-column specific discrimination of ochratoxin A

Talanta 200 (2019) 193–202

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Preparation and evaluation of highly hydrophilic aptamer-based hybrid affinity monolith for on-column specific discrimination of ochratoxin A Yiqiong Chen, Xinyue Ding, Dandan Zhu, Xucong Lin , Zenghong Xie ⁎

T

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Institute of Food Safety and Environment Monitoring, Fuzhou University, Fuzhou 350108, China

ARTICLE INFO

ABSTRACT

Keywords: Hydrophilic Aptamer Hybrid affinity monolith Specific discrimination Ochratoxin A

Nonspecific adsorption is a challenge of specific recognition on aptamer-based affinity monoliths. Here, a novel highly hydrophilic polyhedral oligomeric silsesquioxane (POSS)-containing aptamer-based hybrid-silica affinity monolith with a good recognition nature was prepared and used for specific discrimination of ochratoxin A (OTA). A homogeneous polymerization mixture consisted of POSS chemicals, hydrophilic monomers and aptamer solution was directly polymerized via the “one-pot” method. Preparation and characterization of the resultant affinity monolith were studied in detail. A highly hydrophilic nature was obtained and the typical hydrophilic interaction liquid chromatography (HILIC) was observed when acetonitrile (ACN) content in mobile phase was 25%, which reached the highest hydrophilicity of POSS-based hybrid monoliths. By using OTA as model analyte, the nonspecific adsorption was effectively suppressed. The recovery of the analogue ochratoxin B (OTB) was only about 0.1% even if the content of OTB was 50 times more than OTA, which was much better than other POSS-containing monoliths and polar siloxane-based hybrid monoliths. Applied to beer samples, the adsorption of background materials was drastically resisted, and efficient recognition of OTA was obtained with the recoveries of 94.9–99.8%. Much less disturbance was observed than that occurred in hydrophobic POSS-based affinity monolith. It lights an attractive implement with high hydrophilicity and specificity for online selective recognition of OTA.

1. Introduction Specific sample preparation is critical for the accurate determination of target analyte in real samples. As the alternative and attractive affinity technology, aptamers that possess of their unique merits such as facile modification, good stability and high specificity towards target molecules have caught much attention [1–3]. Currently, aptamermodified monolithic columns (Apt-MCs) have been well studied for the on-column recognition of trace analytes attributing to their merits including high permeability, fast mass transfer, specific affinity and convenient online coupling with analytical techniques [4,5]. Developing high-performance Apt-MCs for the efficient on-column discrimination of target molecule is interesting. To date, many Apt-MCs based on organic polymer monolithic supports or siloxane-based hybrid monolithic supports have been developed. Zhao’s groups put forward aptamer-modified functionalized glycidyl methacrylate (GMA)-based polymer monolith, and the online specific extraction of hydrophobic analytes such as thrombin and cytochrome c was obtained [6,7]. Due to the inherent hydrophobic nature of methacrylate-based monomers, the hydrophobicity of these resultant

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affinity monoliths was notable. Hydrophobic interaction between the analytes and methacrylate-based aptamer-modified monoliths caused an obvious nonspecific adsorption of target analytes on the support materials [8], which led to an inference to the accurate measure of target analytes. The nonspecific adsorption of hydrophobic analytes on methacrylate-based aptamer affinity monoliths always existed, and hydrophobic nature of monolithic supports was one of the critical factors and needed to be improved [8]. On the other hand, hybrid-silica affinity monoliths acting as a rapidly developing functional supports have been employed to immobilize aptamers. Brothier et al. [9] and Chi et al. [10] fabricated aptamer-modified hybrid monoliths by the postcolumn modification of aptamers on siloxane-based hybrid matrix. However, the common commercial alkoxysilanes were often employed to form the precursors and fabricate silica-hybrid affinity monoliths. The rigorous “sol-gel” operation process should be adopted and polar Si-OH groups would be exposed, which might cause an obvious adsorption in the resultant hybrid affinity monoliths. As shown in the typical cases [9,10], the nonspecific adsorption between target OTA and the control column was notable with a recovery of 14.1 ± 9.5%, and cross-reactivity towards OTB exhibited the higher level of

Corresponding authors. E-mail address: [email protected] (X. Lin).

https://doi.org/10.1016/j.talanta.2019.03.053 Received 26 September 2018; Received in revised form 10 March 2019; Accepted 14 March 2019 Available online 14 March 2019 0039-9140/ © 2019 Published by Elsevier B.V.

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18.7 ± 6.0% [9]. So far, though a wide application for the on-line specific recognition of target analytes have been developed with AptMCs, highly hydrophobic nature in acrylate-based polymer monolithic supports or polar Si-OH groups in silica-hybrid monoliths still exist, and the nonspecific adsorption observed on affinity monoliths might be notable and lead to a potential threat to the selectivity [6,8]. Seeking an ideal approach to improve the hydrophilic nature and eliminate polar Si-OH groups on the affinity monoliths would be essential. Polyhedral oligomeric silsesquioxane (POSS) chemicals as the ideal alternative to alkoxysilanes with unique cage-like structures without silanol groups have aroused wide concern [11,12]. By means of a “onepot” strategy, the co-polymerization of POSS chemicals and organic monomers could be effectively completed and the whole co-polymerization reaction was facile to be handled [13,14]. The rigorous hydrolysis and condensation manipulations for alkoxysilanes in the traditional “sol-gel” chemistry and the time-consuming post-column modification could be overcome [15,16]. Lately, an interesting POSScontained aptamer-based hybrid silica monolithic column has been reported in our newly works [17], in which the nonspecific adsorption caused by Si-OH polar groups that prepared with the “sol-gel” process could be avoided. The control on nonspecific adsorption of the analogue OTB was notably improved and the average recovery of OTB was controlled at 5.5 ± 0.3% if the content of OTB was 0.2 ng, which was better than that obtained in the references [9]. It lights a promising approach to preparing the efficient hybrid aptamer-based affinity monoliths. However, commercial POSS chemicals were hydrophobic and hydrophilic nature of the reported POSS-containing affinity monolith was limited. Nonspecific interactions of the analogue OTB and background materials in real samples were still obvious [17]. POSScontaining hybrid monolithic columns were almost highly hydrophobic, and the highly hydrophilic aptamer-modified hybrid affinity monoliths have still not been reported. It is significant to develop a highly hydrophilic POSS-containing Apt-MC with low nonspecific adsorption for on-column discrimination of target molecule. Meanwhile, as well as we known, ochratoxin A (OTA) which acts as the most common mycotoxin produced by Aspergillus and Penicillium moulds, could cause severely adverse effects such as nephrotoxic, immunotoxic, neurotoxic and carcinogenic actions on humans, and should be sensitively detected [18]. Currently, many analytical methods have been developed for sensitive determination of OTA, such as enzymelinked immunosorbent assay (ELISA) [19,20], high performance liquid chromatography (HPLC) coupling with fluorescence detection (FLD) [21,22] or mass spectrometry (MS) [23], and ultra-fast liquid chromatography (UFLC) with tandem mass spectrometry (MS/MS) [24]. Though sensitive detection of OTA has been achieved, the specific sample preparation is still acting as the key step for the accurate analysis. Specific sample preparation of trace OTA in real samples is still important. Therefore, by using ochratoxin A (OTA) as model analyte, a novel highly hydrophilic aptamer-modified POSS-containing Apt-MC was prepared via a facile “one-pot” process. The hydrophilic nature and HILIC mechanism were evaluated in detail and compared with the hydrophobic Apt-MC reported previously. The affinity retention behavior was examined, and the possible nonspecific adsorption of the analogue OTB was also examined. Based on the advantage of high hydrophilicity of the obtained Apt-MC, an excellent stability and high selectivity towards OTA were achieved with low nonspecific hydrophobic interaction. Specific extraction of OTA in beer samples on hydrophilic affinity monolith was demonstrated. The nonspecific adsorption of background materials was drastically decreased and the efficient on-column discrimination of OTA was realized. It provides a potential facile access to preparing highly specific affinity monoliths for on-column discrimination of OTA.

2. Experimental 2.1. Chemicals and materials Polyhedral oligomeric silsesquioxane methacryl substituted (POSSMA, cage mixture n = 8–12), polyethylene glycol (PEG, Mol. wt 10,000) were purchased from Sigma-Aldrich (USA). 2-Acrylamido-2methyl propane sulfonic acid (AMPS, 98%) was purchased from Tokyo chemical industry. N,N′-methylene-bis-acrylamide (MBA, ≥ 99.0%), ochratoxin A (OTA, 98%), ochratoxin B (OTB, 98%) and aflatoxin B1 (AFB1, 98%) were purchased from Aladdin Industrial Co. (Shanghai, China). The chemical structure of OTA (CAS: 303-47-9) was added in Fig. S1. Fused-silica capillary (100 µm i.d. × 365 µm o.d.) was obtained from Yongnian optic fiber plant (Hebei, China). Aptamer targeting OTA (5′-SH-C6-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3′, denoted as Apt36), fluorescence labeled Apt36 (5′-SH-C6-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3′-FAM, denoted as Apt36-3′-FAM) and control oligonucleotide (5′-SH-C6-CTG GCC CAG ATT TTA AGG TGC GTA AAG AAA AAA AGT-3′, denoted as control ssDNA) were fabricated and purified by Sangon Biotech. Co.(Shanghai, China). The binding buffer (BB) consists of Tris-HCl 10 mM, NaCl 120 mM, CaCl2 20 mM, KCl 5.0 mM, pH 8.50, and the elution solvent consists of ACN/TE buffer solution (Tris-HCl 10 mmol/L, EDTA 2.5 mmol/L, pH 8.00) = 30/70 (v/v) were prepared in this work. Water was purified using a Milli-Q apparatus (Millipore, USA). Other chemicals used in this work were of analytical pure grade. 2.2. Preparation of PMAA affinity monoliths Highly hydrophilic affinity monolith poly(POSS-MA-co-MBA-coAMPS-Apt) (denoted as PMAA) was prepared via a facile “one-pot” process. Prior to the preparation of hybrid monoliths, γ-MAPS was used to introduce the vinyl groups onto the inner surface of capillary [25]. According to the recipes (shown in Table 1), hydrophilic monomers such as N,N′-methylene-bisacrylamide (MBA) and 2-acrylamido-2-methyl propane sulfonic acid (AMPS) were directly mixed and co-polymerized with POSS-MA and aptamer (Fig. 1). The mixture was sonicated for 20 min and then introduced into the pre-treated capillary to an appropriate length by a syringe. With both the ends sealed with rubber stoppers, the capillary was put in a thermostatic bath at 55 °C for 12 h to finish the reaction. Finally, the obtained monoliths were flushed with methanol to remove the residual materials and then stored at 4 °C with the BB solution. For comparison, a bare monolith without aptamer modification was prepared by replacing the aptamer with water, and the control monolith was prepared with control ssDNA. The applied length of affinity monoliths in this work was 10 cm. 2.3. General characterization of PMAA affinity monolith Microscopic morphology was examined by using a scanning electron microscope (Nova Nano SEM 230). According to the literatures [26–28], permeability was measured with a LC-20 CE pump (Shimadzu, Japan) and calculated by the Darcy’s law (shown in Electronic Supplementary Information, ESI). Swelling property value (SP) and retention factor (k) of the monoliths were measured and shown in ESI. According to the references [26,28], toluene and thiourea were used as the model marker compounds to study the retention behavior for evaluating the hydrophilic property of monolithic columns. 2.4. Specific recognition of OTA As shown in Fig. S2, the specific discrimination of OTA was accomplished with the resultant PMAA affinity monolith by online

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Table 1 Effect of synthesis parameters on the formation of aptamer affinity hybrid monoliths. No. of columns prepared

Monomer-to-solvent ratio

POSS-MA (wt%) a

MBA (wt%)

1 2 3 4 5 6 7 8

18:82 18:82 18:82 18:82 18:82 18:82 18:82 18:82

5.0% 5.0% 5.0% 5.0% 5.0% 5.0% 2.5% 7.5%

70.0% 70.0% 70.0% 70.0% 70.0% 70.0% 72.5% 67.5%

b

AMPS (wt%)

DMF (wt%)

c

25.0% 25.0% 25.0% 25.0% 25.0% 25.0% 25.0% 25.0%

d

77.5% 75.0% 74.0% 72.5% 70.0% 70.0% 72.0% 75.0%

PEG (wt%)e

Water (containing aptamer) (wt%) f

Permeability g K (× 10−14 m2)

20.0% 20.0% 20.0% 20.0% 20.0% 20.0% 20.0% 20.0%

2.5% 5.0% 6.0% 7.5% 10.0% 12.5% 8.0% 5.0%

10.5 8.53 5.55 3.95 1.23 n/ah 3.78 3.73

a/b/c. Percentage of POSS-MA/MBA/AMPS in the monomer mixture. d/e/f. Percentage of DMF/PEG/water in porogenic solvents. f. The concentration of aptamer solved in the water was 210 μmol/L. g. The permeability was measured by using methanol. The viscosity of methanol was 0.544. n/a: The measurements could not be accomplished.

coupling with HPLC system. A 20-μL of OTA solution dissolved in BB solution was percolated through the monolithic column at a flow of 0.02 mL/min with a constant pressure drop of 250 psi. Then, the affinity monolith was washed with BB solution at a flow of 0.1 mL/min under 500 psi to remove the nonspecific adsorption. Subsequently, the affinity monolith was eluted with the BB solution to ensure no residual OTA signal could be eluted. Then the elution solvent was switched to ACN/ TE solution (ACN/TE = 30/70, v/v) to elute the captured OTA. All the elution fractions (20 μL) were measured by HPLC-FLD system. The chromatographic separation condition was set according the reference [17]. The fluorescence excitation and emission wavelengths were 333 nm and 460 nm, respectively.

2.6. Sample analysis 2.6.1. Specific sample enrichments with PMAA monolith Beer samples with ethanol content of 3.6% were chosen. Usually, there is not the maximum tolerable OTA concentration in beer. Seen from the previous report [32], the actual OTA residue contents reported in beer are often less than 0.20 ng/mL, and so the specific enrichment operation would be employed. In this work, beer samples were prepared similar to the reference reported previously [9]. Briefly, a 10 mL of beer was degassed ultrasonically for 3 h and cooled at 4 °C for 30 min. The pH of beer sample was adjusted to 8.50 with a 2.0 mol/L NaOH solution by using a pH-meter. After being filtered through the 0.22-μm membrane, the beer solution was spiked with OTA at different concentrations ranging from 0.025 to 0.20 ng/mL. Then the fortified beer samples were diluted with BB solution (pH = 8.50) at a ratio of 1:1 (v/v) with the dilution factor of 2. Finally, a 100-μL of the fortified beer solution was employed for five-fold volume enrichment and sample analysis. Each fortified beer sample was measured three times.

2.5. Specificity and nonspecific adsorption To investigate the specificity of the affinity monolith, bare monolith, control ssDNA modified monolith and PMAA monolith were applied to capture OTA respectively with the procedure mentioned in Section 2.4. According to the literatures [29,30], OTB was a typical structural analogue of OTA, and AFB1 was a widespread toxin and might co-exist with OTA [31]. Here, OTB and AFB1 were selected as the model structural analogues and co-existed interferences respectively to evaluate the cross-reaction on these monolithic columns with a similar procedure. To further evaluate the nonspecific adsorption on PMAA monolith, the mixture of OTA and OTB with different content ratios such as 1:1 and 1:50 were further adopted and the adsorption of OTB was evaluated according to the procedure mentioned above.

2.6.2. Sample analysis for HPLC-MS control method According to the previous reports [33], the pretreatment of beer samples for HPLC-MS was preceded by using C18 solid-phase extraction column (500 mg/3 cc, Waters Sep-pak) and the analysis process was shown in ESI. 3. Results and discussions 3.1. Preparation and optimization of PMAA affinity monolith As shown in Fig. 1, PMAA monolith was fabricated via a direct “onepot” process. Highly hydrophilic AMPS and MBA were employed, and

Fig. 1. Scheme on the preparation of PMAA hybrid affinity monolith with “one-pot” process. 195

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Fig. 2. SEM images and affinity recognition of OTA with PMAA monoliths (a-d) Contents of water in porogenic system: (a) 5.0%; (b) 6.0%; (c) 7.5%; (d) 10.0%. Magnification times: (a1, b1, c1, d1) × 700, others × 2000. Peak 0: background peak, 1: OTA (10 ng/mL).

POSS-MA with a rigid framework was employed to reinforce mechanical stability of monolithic columns. According to the reference [17], the ternary porogenic system consisting of H2O/DMF/PEG was selected to form a homogeneous hybrid monolith. As the vital factor of porogenic system, the content of water could cause a significant effect on the porous structure and permeability of POSS-containing hybrid monoliths. As shown in Table 1, with the content of H2O increasing from

2.5% to 10.0% (wt%) in the porogenic system, the permeability of hybrid monoliths decreased obviously from 10.5 × 10−14 m2 to 1.23 × 10−14 m2 (columns No. 1–5, in Table 1). As shown in Fig. 2, the PMAA monoliths were homogeneous, porous and well attachment to the capillary inner wall. Furthermore, numerous through-pore and micropore were observed in the monolithic stationary phase, and polymer clusters with different particle sizes were formed. With the 196

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Fig. 3. Plots of the retention factors (k) versus ACN content (v/v) in different PMAA affinity monoliths (a-c) and PEAA monolith (d) Contents of POSS-MA in PMAA affinity: (a) 2.5%; (b) 5.0%; (c) 7.5%. Experimental conditions: mobile phase, 5 mM phosphate buffer containing ACN from 20% to 90% at pH 6.0; pump flow: 0.1 mL/min; applied pressure: 500 psi.

content of H2O increasing, the average particle sizes of polymer clusters in monolithic columns (columns No. 2–5, in Table 1) were changed smaller and estimated in the range of 5.4–10 µm, 4.2–6.0 µm, 2.0–4.0 µm and 1.0–2.0 µm, respectively. It might be attributed to the suppression caused by H2O inhibitor on the free radical polymerization of polymer cluster and resulted in an obvious decrease of the permeability in PMAA monoliths. When used for the specific extraction, the obvious fluorescence signal of OTA could be observed in these PMAA affinity monoliths (Fig. 2, a3-d3). It gradually increased with the permeability of monolithic columns decreasing and reached the highest value when the permeability was 3.95 × 10−14 m2 (column No. 4 in Table 1, Fig. 2-c-3). The proper content of water in porogenic system was critical to gain a suitable permeability of PMAA monoliths for a fast mass transfer and specific recognition of OTA. The optimization of POSS-MA for achieving a highly hydrophilic nature of PMAA monolith was studied. With the content of POSS-MA increasing from 2.5% to 7.5% (columns No. 7, 4, 8, in Table 1), the permeability was similar as about 3.78 × 10−14 m2, while the hydrophilic property changed significantly. Additionally, the mechanism of hydrophilic interaction (HI) was also evaluated by the plot of k (retention factor) vs. ACN content in the mobile phase. Usually, the change of retention mechanism from HILIC to RP mode could be observed, and this critical composition of mobile phase corresponding to the inflection point could be used to assess the hydrophilicity of stationary phase [34,35]. As shown in Fig. 3, by increasing the ACN content in mobile phase, the retention factor k of the model marker analytes (toluene and thiourea) was investigated and used to evaluate the hydrophilicity interaction (HI) of PMAA monoliths. A typical HILIC retention behavior was observed with the critical content of ACN rising from 20% (Fig. 3a), 25% (Fig. 3-b) to 31% (Fig. 3-c), respectively. The critical mobile

phase composition in these PMAA monoliths was close to that of highly hydrophilic acrylate-based non-affinity monoliths (about 25%) reported previously [36], and rather less than that of the hydrophobic POSS-based affinity monolith (about 80%) [17] (Fig. 3-d) or other POSS-based hybrid non-affinity monoliths (about 60%) [37,38]. A high hydrophilicity could be achieved in PMAA monoliths with the content of POSS-MA ranging from 2.5% to 7.5%. To further explore the influence of POSS-MA on the resultant PMAA monoliths, two factors such as swelling properties value (SP) and adsorption of OTB were evaluated. As shown in Table 2, with the content of POSS-MA increasing in 2.5–7.5%, the recovery yields of OTA were ~93%. However, the recoveries of OTB were only 0%, 0.1% and 3.9% respectively, which were much less than the data of 7.5% obtained on the hydrophobic POSS-containing aptamer-based affinity hybrid monolith [17]. PMAA monolithic columns with high hydrophilicity could effectively resist the nonspecific interaction. But it was notable that, SP values of these PMAA monoliths were −0.64, −0.48 and Table 2 Affinity analysis on three hybrid affinity monoliths with different hydrophilicity. Affinity column

No. of columns prepared

Recovery of OTA (%)

Recovery of OTB (%)

SP (THF)

Hydrophilic PMAA monolith

7 4 8 –

92.2 93.4 93.6 93.7

0 0.1 3.9 7.5

−0.64 −0.48 −0.40 0.40

Hydrophobic PEAA monolith

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bare monolithic column and control monolithic column. For the PMAA monolithic column, OTA can efficiently bind with aptamer to form the adapted conformation in the presence of divalent cations with BB solution. With the aid of the carboxyl and 8-hydroxyl groups in OTA, a bridging interaction could be mediated by OTA coordination complex with Ca2+ cation (binding constant: logK(OTACa) = 2.78) [40], and enhanced the binding of OTA to the aptamers [29]. No obvious OTA was observed in the percolation fraction (as shown in Fig. 5-c1). After the mobile phase was switched to ACN/TE elution solution, EDTA was employed to chelate Ca2+ cation with a larger binding constant {logK(EDTA-Ca) = 10.20}, thus the OTA-Ca complex could be disrupted by EDTA and OTA was eluted by ACN from PMAA monolith. As seen from Fig. 5-c3, a notable response of OTA was detected in the ACN/TE elution solution and the recovery of OTA reached 92.1 ± 1.8% (n = 3), which indicated that OTA was specifically captured and eluted with PMAA monolith.

Fig. 4. Effects of the content of aptamer on fluorescence response of OTA.

3.2.3. Stability and repeatability The mechanical stability of PMAA monolithic column was evaluated with different mobile phases. A good mechanical stability was achieved with an excellent linear relationship (R2 > 0.9973) between the backpressure and flow rate of different mobile phase such as water, ACN and methanol. The PMAA affinity monolith was applied to the specific recognition of OTA (10 ng/mL). In the ACN/TE elution solvent, OTA could be well detected. The average recovery yields of OTA on PMAA affinity monoliths were gained at about 92%, and the intra-day and inter-day RSDs were also gained at 1.5% and 2.0% (n = 3), respectively. The PMAA hybrid monoliths could be used more than 30 times in two months with an acceptable recovery more than 84.5 ± 1.7%. The good stability and repeatability for specific extraction could be carried out on PMAA affinity monoliths.

−0.40 when the contents of POSS-MA were of 2.5%, 5.0% and 7.5%, respectively. With the content of POSS-MA decreasing, the hydrophilic nature of PMAA affinity monoliths increased and specific adsorption property enhanced, while the swelling/shrinkage influence (SP value) would aggravate. Besides, the amount of aptamer in the preparation mixture was optimized. As shown in Fig. 4, the greatest response of OTA captured by PMAA affinity monoliths was obtained at the concentration of aptamer of 210 μmol/L. In summary, via the “one-pot” process, highly hydrophilic PMAA hybrid affinity monolith could be efficiently established, and the column (No. 4, in Table 1) with homogeneous structure morphology (Fig. 2-c) and proper permeability could be chosen as the optimal for the further affinity interaction with OTA.

3.3. Specificity of PMAA monolith

3.2. Characterization of PMAA hybrid affinity monolith

To evaluate the specific adsorption nature on PMAA monolith, a toxin mixture including OTA, OTB and AFB1 was chosen, and the result was shown in Fig. 6. Three toxins could be obviously detected in both the percolation fraction and washing fraction of bare monolith (Fig. 6a1, a2) or control monolith (Fig. 6-b1, b2), respectively. No target analytes were found in the ACN/TE elution solvent (Fig. 6-a3, b3). It indicated that the toxins could not be effectively captured by both the bare column and control monolithic column. On the contrary, as shown in Fig. 6-c1~c2, no obvious signal of OTA was detected in the percolation and washing fractions, while almost all OTB and AFB1 could penetrate through PMAA affinity monolith and the rest was cleaned out in the latter washing procedure. After switching to the ACN/TE elution solution, an impressive response of OTA was achieved (Fig. 6-c3), which demonstrated that OTA could be specifically captured with hydrophilic PMAA monolith. To further illuminate the specific adsorption nature of PMAA monoliths, the retentions and recoveries of OTA and OTB were quantitatively measured. By coupling with HPLC-FLD system, the discrimination of OTA and OTB in different samples was performed by using PMAA monolith, even in the mixture sample with a high content of OTB with a ratio of OTA/OTB up to 1:50 (c/c). As shown in Table 3, after an enough washing the PMAA monolith with the BB solution, the analogue OTB could be efficiently removed from highly hydrophilic monolith, while OTA could be captured and detected with the high recovery yields up to 91.1 ± 2.3%–93.4 ± 2.3% (mean ± SD, n = 3) for different concentrations (0.2–10 ng/mL), while the recoveries of OTB with the same content were at 0–0.1 ± 0.1% (n = 3). Particularly, in the mixture sample with a high ratio of OTA/OTB up to 1:50 (c/c), the average recovery of OTA (0.2 ng/mL) on PMAA monolith reached 92.2 ± 2.5% (n = 3) while the recovery of OTB with a high content up to 10 ng/mL was only 0.1 ± 0.2% (n = 3). According to the literatures [9,17] with hydrophobic monoliths or

Basic characteristics such as morphology, hydrophilic nature of PMAA hybrid affinity monoliths have been well studied in the preparation section mentioned above (Fig. 2 and Fig. 3). Here, other important characteristics including aptamer reaction efficiency, specific recognition, column stability and repeatability were also evaluated in detail. 3.2.1. Aptamer reaction efficiency The fluorescence-labeled aptamer (Apt36-3′-FAM, 210 μmol/L) was used to prepare fluorescent aptamer-modified monolithic column. After an enough washing procedure to remove the unbound Apt36-3′-FAM, the amount of Apt36-3′-FAM immobilized on the monolithic column was determined by measuring the concentration decrease of the Apt363′-FAM solution before and after the “one-pot” process. As shown in Table S2, the effective immobilization percentage of Apt36-3′-FAM in monolithic column was gained at 81.9% (n = 3), which was slightly better than the value of 78% in siloxane-based aptamer-based affinity monolith [39], or similar to the value of 84% on other POSS-containing hybrid affinity monolith [17]. 3.2.2. On-column specific recognition To investigate the specificity of PMAA monoliths towards OTA, three different monolithic columns included PMAA monolith, bare monolith and control monolith were employed. The fractions resulting from three functional monoliths were detected with HPLC-FLD system and shown in Fig. 5. An obvious signal of the OTA was effectively detected in the percolation fraction and washing fraction in the bare monolithic column (Fig. 5-a1, Fig. 5-a2) and control monolithic column (Fig. 5-b1, Fig. 5-b2), respectively. No obvious OTA was eluted and detected (Fig. 5-a3, Fig. 5-b3) after the mobile phase was switched to ACN/TE elution solution. OTA could not be specifically retained on the 198

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Fig. 5. Chromatograms showing specific recognition of OTA with different monoliths (a) bare monolith (unmodified aptamer); (b) control monolith (prepared with control ssDNA); (c) PMAA monolith (prepared with Apt36). a1–c1: percolation fractions; a2–c2: washing fractions; a3–c3: elution fractions The binding buffer and ACN/TE elution solvent were used as the mobile phases before and after the switch point, respectively. Peak 0: background response of binding buffer or elution solvent, 1:OTA (10 ng/mL).

polar silica-hybrid monoliths with Si-OH groups, a relatively serious adsorption of OTB was detected. The recovery of OTB (the content was 0.2 ng) in the reported affinity monoliths such as poly(TEOS-co-APTES) @aptamer, POSS-PEI@AuNPs@aptamer and hydrophobic PEAA control monolith were shown as 18.7% [9], 8.3% [41], and 7.5% [17] (n = 3) respectively, which was hundreds of folds more than that of hydrophilic PMAA monolith. PMAA monolith could significantly resist the adsorption of the analogue and possess better discrimination ability towards target analyte.

3.4. Application A series of OTA standard solutions with the concentrations (0.06–5.0 ng/mL) were measured for evaluating the theory detection limit of OTA on the PMAA affinity monolith. As shown in Fig. 7, the limit of detection (LOD) and the limit of quantitation (LOQ) of OTA in the standard solutions were measured and obtained at 0.06 ng/mL (S/ N = 3) and 0.10 ng/mL (S/N = 10), respectively (shown in Fig. 7). The prepared PMAA affinity hybrid monolith presents a highly specific

Fig. 6. Chromatograms showing the cross-reactivity of three toxins on different monoliths (a) bare monolith; (b) control monolith; (c) PMAA monolith. Peak 0: response of background compounds, 1:OTA, 2:OTB, 3:AFB1. The concentration of each toxin was 10 ng/mL (the content of each toxin was 0.2 ng). Other conditions were the same as in Fig. 5. 199

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Table 3 Recoveries for the selective recognition of OTA by using PMAA affinity monolith. OTA (ng/mL)

0.20 1.00 10.0 0.20

OTB (ng/mL)

0.20 1.00 10.0 10.0

Recovery of OTA (%)

Recovery of OTB (%)

Mean (n = 3)

SD

Mean ( n = 3)

SD

91.1 92.1 93.4 92.2

2.3 3.3 2.3 2.5

– – 0.1 0.1

– – 0.1 0.2

Table 4 Analysis of beer samples by using HPLC-FLD with PMAA monolith or HPLC-MS. No.

1 2 3 4

OTA spiked concentrations (ng/mL) Blank 0.05 0.08 0.10

HPLC-MS

This work

Recoveries (%) (n = 3)

RSD% (n = 3)

Recoveries (%) (n = 3)

RSD% (n = 3)

– 104.5 ± 1.9 100.8 ± 3.2 97.5 ± 2.8

– 1.8 3.2 2.8

– 94.9 ± 2.8 95.9 ± 2.5 99.8 ± 2.7

– 2.9 2.6 2.7

-: Unable to be detected.

SD: the abbreviation of standard deviation. -: Unable to be detected.

of beer samples, while a gentle chromatography and baseline could be obtained with the resultant PMAA monolith. The background signal response in the PMAA monolith was only 1/10 or more lower to that of hydrophobic PEAA monolith. The results indicated the nonspecific adsorption of background compounds on the PMAA monolith could be drastically resisted, which was favorable for the accurate quantification of target OTA. To express the specific recognition nature, the limit of detection (LOD) and the limit of quantitation (LOQ) of OTA in the fortified beer samples were also examined. As shown in Fig. 8-c~d, the OTA could be selectively extracted from the complicate background signals by using the PMAA affinity monolith, and the LOD and LOQ of OTA in the beer samples reached at 0.025 ng/mL (S/N = 3) and 0.05 ng/mL (S/ N = 10), respectively. Used for the beer samples spiked with different levels of OTA as 0.05, 0.08 and 0.10 ng/mL, the satisfactory recoveries of fortified OTA samples were measured as 94.9 ± 2.8%, 95.9 ± 2.5% and 99.8 ± 2.7% (n = 3), respectively. To further verify the detection accuracy of the HPLC-FLD method developed with PMAA affinity monolith, a typical LC-MS method was employed. As shown in Fig. S3a, no obvious OTA was detected in the blank beer samples. With the spiked concentrations of OTA increasing, OTA could be detected and the peak area of OTA was increased (Fig. S3-b-d). As shown in Table 4, the recoveries of OTA were obtained at 97.5 ± 2.8%–104.5 ± 1.9% (n = 3) for the spiked levels of OTA as 0.05, 0.08 and 0.10 ng/mL respectively. The result of PMAA affinity monolith coupling with HPLCFLD was acceptable, and there's not much difference in the recoveries between this method and LC-MS. Besides, in comparison with other previous reports [9,10,17,41], as shown in Table 5, the specific recognition and recovery towards OTA were better than that reported previously and seem acceptable. As a result, the highly hydrophilic PMAA monolith could significantly reduce the adsorption of background materials and facilitate the on-column specific discrimination of trace OTA in beer samples.

Fig. 7. Detection limit of OTA with PMAA monolith in HPLC-FLD system.

capture of OTA with a sensitive detection limit that could be used for specific extraction of trace OTA. Applied to beer samples, the discrimination of OTA with the hydrophilic PMAA monolith was investigated. The actual OTA residues reported in beer samples are less than 0.20 ng/mL [32], therefore 5folds volume enrichment was employed to improve the analysis sensitivity of OTA, and the results were shown in Fig. 8. In Fig. 8-a, by using the hydrophobic PEAA monolith, a rather serious fluorescence response of background compounds eluted by the ACN/TE eluent was detected in the blank beer sample, while the interference phenomenon observed in hydrophilic PMAA monolith (shown in Fig. 8-b, red line) was significantly reduced. A serious interference signal were observed when hydrophobic PEAA monolith was employed for the specific extraction

Fig. 8. Detection of trace OTA in beer samples with the PEAA affinity monolith (a) and PMAA affinity monolith (b-d). Spiked OTA (ng/mL): (a, b) 0; (c) 0.025; (d) 0.050. Peak 1: OTA. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article) 200

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Table 5 Comparison of the specific analysis of OTA in this work with that of previous reports. Affinity material

Spiking level (ng/mL)

Recovery (%)

LOD/LOQ (ng/mL)

Repeatabilitye (RSD%)

Reproducibilityf (RSD%)

Ref.

Aptamer-modified siloxane hybrid monolith AuNPs@aptamer-modified siloxane hybrid monolith POSS-PEI@AuNPs@aptamer hybrid monolith POSS containing hydrophobic PEAA affinity monolith POSS containing hydrophobic PMAA affinity monolith

20~300 0.5~5.0

≥ 80 88.6–94.1

–/– 0.025/–c

–/– 2.0/2.4

< 10.0 2.6

[9] [10]

2.0~25.0 0.05~0.10

92.5–94.5 92.7–101.2a

1.9/2.8 1.9/3.2

2.2 3.4

[41] [17]

0.05~0.10

94.9–99.8b

0.06/0.1c 0.05/0.1c 0.025/0.045d 0.06/0.1c 0.025/0.05d

1.5/2.0

2.5

This work

–: the data was not provided in the reference. a The recoveries were obtained from the chromatograms with a manual revision. b The recoveries were obtained directly from the chromatograms. c LOD and LOQ were calculated in standard sample. d LOD and LOQ were calculated in real sample. e The repeatability was examined through the intra-day and inter-day RSD of the recoveries of OTA. f The reproducibility was examined through the RSD of the recoveries of OTA by using three batches of affinity monoliths.

4. Conclusions

References

A novel highly hydrophilic POSS-containing aptamer-modified hybrid affinity monolith with a good specific adsorption nature was presented via a facile “one-pot” process. A highly hydrophilic nature was achieved when the ACN content in mobile phase exceeded 25%, which was much better than that of POSS-based monoliths (about 60–80%) reported previously. Highly specific extraction of OTA was achieved with the extremely weak of cross-reactivity towards OTB and AFB1. Particularly, trace OTA (0.2 ng/mL) can be well recognized even in the mixture with a high content of OTB (OTA:OTB = 1:50). High specificity towards OTA was carried out with a weak recovery of OTB only about 0.1%, which was much better than that of 7.5% obtained in hydrophobic POSS-containing affinity monolith, 8.3% in AuNPs@aptamer modified POSS-PEI affinity monolith and 18.7% in polar silica-hybrid affinity monolith. Applied to beer samples, the nonspecific adsorption of background compounds on the result PMAA monolith was drastically inhibited. Better baseline and retention chromatograms could be obtained by using PMAA monolith, while a more serious baseline drift happened on the hydrophobic PEAA which was reported previously and used as the control POSS-containing monolith here. With 5-folds volume enrichment, the sensitive LOD of OTA in beer samples reached 0.025 ng/mL. The satisfactory recoveries were gained at 94.9 ± 2.8%, 95.9 ± 2.5% and 99.8 ± 2.7% (n = 3) with the concentration of 0.05–0.10 ng/mL. It provides an access to facilely fabricating a highly hydrophilic POSS-containing aptamer-based hybrid affinity monolith for the efficient discrimination of OTA with low nonspecific adsorption.

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