Fang Songa,
Zhexiang Zhangb,
Xuerong Xu*a and
Xucong Lin*b
aCollege of Economics and Management, Fujian Agriculture and Forestry University, Fuzhou 350001, China. E-mail: xxrfafu@163.com
bEngineering Technology Research Center on Reagent and Instrument for Rapid Detection of Product Quality and Food Safety in Fujian Province, Fuzhou University, Fuzhou, 350108, China. E-mail: linxucong@aliyun.com
First published on 20th October 2023
Enabling cost-effective safety monitoring of shellfish is an important measure for the healthy development of the coastal marine economy. Herein, a new aptamer@metal–organic framework (MOF)-functionalized affinity monolithic column was proposed and applied in selective in-tube solid-phase microextraction (IT-SPME) coupled with HPLC for the accurate recognition of domoic acid (DA) in shellfish. Using a surface engineering strategy, ZIF-8 MOF was grown in situ inside the poly(epoxy-MA-co-POSS-MA) hybrid monolith. A high BET surface area and abundant metal reactive sites of the MOF framework were obtained for anchoring massive aptamers with terminal-modified phosphate groups. Various characterizations, such as SEM, elemental mapping, XRD, and BET, were performed, and the affinity performance was also studied. The presence of a massive amount of aptamers with a super coverage density of 3140 μmol L−1 bound on ZIF-8 MOF activated a high-performance bionic-affinity interface, and perfect specificity was exhibited with little interference of tissue matrixes, thus assuring the highly selective capture of DA from the complex matrixes. Under the optimal conditions, DA toxins in shellfish were detected with the limit of detection (LOD) of 7.0 ng mL−1 (equivalent to 14.0 μg kg−1), representing a 5–28 fold enhancement in detection sensitivity over traditional SPE or MIP adsorbents reported previously. The recoveries of fortified mussel and clam samples were achieved as 91.8 ± 1.2%–94.1 ± 1.9% (n = 3) and 91.2 ± 1.1%–94.5 ± 3.6% (n = 3), respectively. This work sheds light on a cost-effective method for online selective IT-SPME and the accurate monitoring of DA toxins using an aptamer@MOF-mediated affinity monolith system coupled with the inexpensive HPLC-UV technique.
Currently, HPLC-UV detection with automatic sampling units has been developed and widely used for quantifying the DA toxin.5 Due to the low content of DA in natural shellfish with complicated matrixes, it is hard to directly achieve the precise quantitation of DA by common HPLC-UV methods.6,7 Pretreatment is necessary, and therefore, developing an efficient recognition strategy that can be online integrated with HPLC for the automatic analysis of DA with good selectivity and sensitivity would be significant. Some common methods such as the traditional liquid–liquid extraction (LLE)8,9 and solid-phase extraction (SPE)10 have been reported for the DA extraction. As the most popular extraction techniques, SPE phases, such as ion-exchange resins,11 amorphous titania,12 molecularly imprinted polymer (MIP),13,14 or C18 reversed-phase polymer,15 have been demonstrated for the purification of DA in shellfish. Magnetic solid-phase extraction (MSPE) was also developed for the isolation of DA using UiO-66 modified magnetite@silica microspheres as the sorbents.16 Though the traditional SPE and MSPE techniques are currently widely adopted and exhibit effective extraction performance, there still are problems to be resolved, such as high solvent consumption, high particle dispersity, and unsatisfactory selectivity.6,8,17 Further, these methods are usually used in an offline manual way and hardly online coupled with LC for automatic analysis,6,18 resulting in a number of issues, such as non-automated operation, multiple steps, analyte loss, long-time operation periods, and low efficiency. Improvements that have attached attention including attempts to automate the techniques, since SPE and MSPE processes generally require offline procedures (loading, washing, elution) and do not seem to be particularly able to cope with batch samples. In particular, the selectivity in traditional SPE and MSPE methods toward the target DA toxin is limited, and certain analogues could be co-adsorbed, resulting in a non-negligible unspecific adsorption and obvious background interference.
As for the other issues, in-tube solid-phase microextraction (IT-SPME), as an emerging extraction protocol, has been proposed and even applied as a powerful and reliable tool for online sample pretreatment, which integrates SPME with HPLC for automatic analysis.19,20 This can effectively reduce most the problems that occur in the offline mode, while the system also exhibits other merits, such as options for miniaturization, automation, and green characteristics.21 Using a functional adsorbent, selective IT-SPME methods have been proposed as an ideal online sample preparation technique, opening up effective access to the automatic identification of a target analyte.22 Recently, using a MIP monolith, a specific recognition strategy of domoic acid was explored to accomplish selective IT-SPME.23 Various advantages, such as skilled preparation, physicochemical robustness, and reusability for DA recognition, were achieved; however, it also suffers from some issues, such as the inevitable template bleeding, tedious elution process, high capital expenditure, and low conversion efficiency. Consequently, there is still a requirement to further develop a cost-effective and specific means for the online recognition of DA in shellfish.
Aptamers, as inexpensive nucleic acid molecules with a flexible structure, and good biocompatibility, which can fold into 3D shapes and bind to specific targets, possess high stability and selectivity to target molecules and have been developed for the special analysis of target analytes.23,24 As a new bionic material, aptamer-based affinity monolithic columns with various unique merits, such as high tolerance, fast mass transfer, good affinity performance, and convenient coupling with instruments,25 have been widely designed as a promising approach with a “molecular recognition” mechanism for selective IT-SPME for the online specific recognition of various analytes.26 Indeed, various methacrylate-based organic polymer monoliths and polyhedral oligomeric silsesquioxane (POSS)-based hybrid affinity monoliths modified with aptamers have been reported and used for the IT-SMPE of trace contaminants (e.g., mycotoxins, BPA, MC-LR).27–29 Using gold nanoparticles (AuNPs) as a medium, aptamer@AuNPs-functionalized monoliths with a high Brunauer–Emmett–Teller (BET) surface area and large content of aptamers have also been designed for perfect specific performance.30–32 The selective IT-SPME of the massive amount of aptamers on the affinity monolith offers an attractive opportunity for the online selective capture and precise measurement of various analytes.33 This sheds light on a promising approach for the online accurate recognition of DA. So far, aptamer-based affinity materials have received much attention, while online selective IT-SPME for the accurate analysis of DA based on affinity monoliths is still absent.
Inspired by the above facts, a novel aptamer@metal–organic framework (aptamer@MOF)-functionalized monolith with massive aptamers is proposed and was adopted for selective IT-SPME for the online specific recognition of DA (Fig. 1). Using monomers such as Epoxy-MA and POSS-MA, multiple epoxy groups could be introduced on the stereostructure, and thus the resultant poly(epoxy-MA-co-POSS-MA) was designed as a matrix possessing abundant epoxy groups for effectively tethering aminoimidazole molecules and subsequently growing ZIF-8-MOFs. Meanwhile, ZIF-8 MOFs possessing perfect surface chemistry and sufficient Zn2+ ions were employed for chelation with phosphate to immobilize the aptamers. Attributed to a large surface area and abundant Zn2+ active sites, massive amounts of aptamers could be efficiently anchored at the MOF sites in the monolith. The high coverage density of aptamers modified in the affinity column could enhance the binding capacity or specific recognition ability. Thus, a mass of affinity interaction sites were engineered, which enabled the online specific bio-recognition of DA. Herein, various characterizations, such as SEM, elemental mapping, XRD, and BET, were performed, and the performance of the aptamer@MOF-modified affinity monolith was verified. The as-developed method was applied for the online recognition of DA in shellfish samples. As result, it sheds light on a promising method for selective IT-SPME for the online identification of DA toxins utilizing a facile HPLC method.
A homogeneous prepolymer mixture consisting of Epoxy-MA (35.00 mg), POSS-MA (35.00 mg), 1-propanol (49.40 mg), PEG-400 (80.60 mg), and AIBN (2.00 mg) was made with vortex oscillation for 3 min and then debubbled under ultrasonic treatment for 15 min. The solution was injected into the pretreated capillary to a 10 cm length with a syringe, which was then sealed at both ends and thermostatically heated at 60 °C in the water bath for reaction for 18 h. The poly(epoxy-MA-co-POSS-MA) monolith (poly(EMPM) matrix monolith) was finally obtained and rinsed with methanol.
Next, 200 μL of 1-(3-aminopropyl)imidazole solution (1 mol L−1) was pumped into the above MP parent monolith, and then the epoxy-ring-opening reaction proceeded in a water bath at 65 °C for 12 h. After rinsing with methanol (10 MPa), 20 μL of Zn(NO3)2 methanol solution (20 mmol L−1) was pumped into the imidazole-modified monolith and held there for 30 min to achieve the modification with Zn2+ ions. Then, 20 μL of dimethylimidazole methanol solution (20 mmol L−1) was pumped into the imidazole-Zn2+-modified monolith and held there for 30 min for the in situ growth of ZIF-8 MOF. The process of zinc nitrate and dimethylimidazole modification was repeated until the self-assembly of the ZIF-8 in the monolithic column was achieved. The obtained monolith was rinsed with methanol.
Finally, 20 μL of aptamer solution (100 μM) was pumped into the ZIF-8-modified monolith using Tris–HCl buffer solution (pH 7.50) as the mobile phase at 0.05 mL min−1. The Apt@ZIF-8 affinity monolith was finally achieved. Additionally, a control monolithic column was prepared with control ssDNA following the same procedure.
The dynamic binding capacity of DA was calculated via the equation shown below. Here, 60 ng mL−1 of DA solution was used to evaluate the aptamer binding capacity, and 20 μL aliquots of the eluent were collected for DA analysis to draw the breakthrough curve.
Qmax = C(VR − V0) |
For a suitable monolithic column, the optimal recipe of poly(EMPM) was evaluated and their performance assessed. As can be seen from Fig. S2,† with the content of monomers increasing, the polymer phase inside the capillary remained homogeneous, while the distribution of polymerization clusters became compact. The permeability of the monolithic column gradually decreased from 12.19 × 10−14 m2 to 0 (Table 1, a–c). As the proportion of crosslinking agent POSS-MA decreased from 22.5% to 12.5%, the crosslinking degree of the polymerization in the monolithic column decreased (Table 1, d–g). The sizes of the polymerization clusters became significantly smaller (in Fig. S3†), while the porosity in the polymerization phase became more obvious, with increasing permeability from 1.27 × 10−14 m2 to 14.63 × 10−14 m2. The as-prepared monolithic column with a proper permeability (6.87 × 10−14 m2) was the optimal and selected as the matrix for the consequent in situ growth of the MOF.
Column designation | Monomer-to-solvent ratio | Epoxy-MAa (%, w/w) | POSS-MAa (%, w/w) | Propanolb (%, w/w) | PEG-400 (%, w/w)b | Cycle of MOF growthc (n) | Permeability Kd (×10−14 m2) |
---|---|---|---|---|---|---|---|
a The percentages of POSS-MA, and Epoxy-MA in the monomer mixtures, respectively.b The percentages of two porogenic reagents in porogenic solvents, respectively. Porogenic reagents were composed of propanol and PEG-400.c The n refers to the number of in situ growth cycles of ZIF-8 MOF in the column.d The permeability was measured using methanol. The viscosity of methanol was 0.544. | |||||||
a | 25:75 | 12.5 | 12.5 | 28.5 | 46.5 | — | 12.19 |
b | 35:65 | 17.5 | 17.5 | 24.7 | 40.3 | — | 2.08 |
c | 45:55 | 22.5 | 22.5 | 20.9 | 34.1 | — | 0 |
d | 35:65 | 12.5 | 22.5 | 24.7 | 40.3 | — | 0 |
e | 35:65 | 15.0 | 20.0 | 24.7 | 40.3 | — | 1.27 |
f | 35:65 | 20.0 | 15.0 | 24.7 | 40.3 | — | 6.87 |
g | 35:65 | 22.5 | 12.5 | 24.7 | 40.3 | — | 14.63 |
h | 35:65 | 20.0 | 15.0 | 24.7 | 40.3 | 2 | 5.93 |
i | 35:65 | 20.0 | 15.0 | 24.7 | 40.3 | 4 | 5.21 |
j | 35:65 | 20.0 | 15.0 | 24.7 | 40.3 | 6 | 4.69 |
k | 35:65 | 20.0 | 15.0 | 24.7 | 40.3 | 8 | 4.26 |
l | 35:65 | 20.0 | 15.0 | 24.7 | 40.3 | 10 | 2.98 |
The optimization of the MOF self-assembly process of the affinity monolithic columns was further demonstrated. As shown in Table 1, h–l, the permeability of the MOF-modified monolithic column decreased from 6.87 × 10−14 m2 to 2.98 × 10−14 m2 with the increase in the number of MOF self-assembly cycles. The response of DA significantly increased after online affinity recognition with the aptamer@MOF-coated monolithic column and reached a maximum at 93.5% with the cycle number of MOF self-assemblies of 8 (in Fig. S4†). Based on the optimization, the aptamer@MOF-modified hybrid-silica monolithic column was achieved and was found to be favourable as an ideal functional medium for the further online specific identification.
In Fig. 3(I-c), it can be seen that the infrared absorption of Zn–N at 423 cm−1 was decreased in comparison to that in Fig. 3(I-b), indicating that the binding of phosphate in the aptamer with Zn2+ occurred and the aptamers were bound on the ZIF-modified monolith. These results indicate that ZIF-8 MOF was successfully synthesized on the monolithic columns. With the modification of the aptamer, the strength of the CN– and Zn–N peaks were weakened in the aptamer@MOF-modified monolith (Fig. 3(I-c)).
The distinct X-ray diffraction (XRD) peaks illustrated the good crystalline morphology in both the ZIF-8 MOFs and aptamer@ZIF-8 modified monoliths (Fig. 3(II-b and c)). A broadened peak band was observed in the poly(epoxy-MA-co-POSS-MA) monolith, suggesting the polymer phase was amorphous (Fig. 3(II-a)). Diffraction peaks were observed mainly distributed at 7.3°, 10.36°, 12.72°, 18.04°, and 24.52° in the aptamer@ZIF-8 modified monolith and were ascribed to the (011), (002), (112), (222), and (233) crystalline planes, respectively. The XRD pattern of the ZIF-8-MOF@polymer monolith was closely consistent with that of the ZIF-8 MOF, indicating that the in situ growth of ZIF-8 MOF had been successfully achieved without damaging its skeleton integrity and crystallinity.
In Fig. S5,† a high thermogravimetric stability of ZIF-8 MOF and poly(EMPM) monolith could be observed before 300 °C, which was consistent with the literature.37 A slight weight loss of less than 4.1% was exhibited before 120 °C, due to the moisture volatilization from air-drying the aptamer@ZIF@poly(EMPM) monolith. Obvious weight losses occurred at 120–335 °C and 335–500 °C, which were mainly caused by the dissociation of the aptamers and ZIF-8@poly(EMPM). The thermal stability of aptamer@ZIF@poly(EMPM) could be maintained at a room temperature of 25 °C to allows its use for in-tube SPME.
In Fig. 3(III), it can be seen that a high BET specific surface for ZIF-8-modified monolith was achieved at 361.50 m2 g−1, which was rather higher than that of the poly(epoxy-MA-co-POSS-MA) monolith (7.18 m2 g−1) or silsesquioxane-based hybrid monoliths (1.1–59.3 m2 g−1).38 This phenomenon indicated that ZIF-8 MOF composed of imidazole groups and zinc ions were anchored on the surface of the monolithic column. The high BET nature and surface ionization offered by the ZIF-8 MOFs were favourable for providing a support matrix and enough reactive sites for a better coverage density of aptamers with negatively charged 5′-terminal phosphate groups.
In Fig. 3(IV), the mechanical stability of the aptamer@MOF modified hybrid monolith was also evaluated under different mobile phases. Linear correlation coefficients between the back pressure and the flow velocity were obtained with the coefficient of determination (R2) above 0.9970. The as-prepared aptamer@MOF-modified monolith illustrated a good mechanical stability in various mobile phases, including methanol, Tris–HCl buffer, water, and the TE elution solution.
To further evaluate the selectivity of the as-prepared aptamer@MOF monolith, the cross-reactivity was investigated by using tryptophan as the classical structural analogue. For the ssDNA control monolith, the analytes were almost in the outflow while no signals were seen in the eluent (Fig. S7a1 and a2†), suggesting that there was no effective retention of DA and tryptophan. Using the ZIF-8 MOF-modified monolith, DA and tryptophan were effectively adsorbed while no analytes could be effectively eluted, with no effective signals observed in the outflow and eluent when using the ZIF-8 MOF-modified monolith (Fig. S7b1 and b2†). When using the aptamer@MOF affinity monolith, an excellent recovery value of DA (up to 94.4%) and negligible detection of tryptophan (nearly 0% of tryptophan) were achieved (Fig. S7c1 and c2†), which indicated that the DA target could be selectively recognized by the aptamer@MOF affinity monolith even with the co-existence of tryptophan. The selectivity of the aptamer-based monolith towards DA toxin was verified showing it was capable of the specific recognition.
Besides, the lifetime of the aptamer@MOF@poly(EMPM) monolith was also estimated (in Fig. 4d). The recovery yields were in a satisfactory stable state and could be maintained at a high level of more than 90% after being consecutively used over 30 days, indicating that the aptamer@MOF@poly(EMPM) monolith was rather stable and serviceable.
Sample | DA spiked in samples (ng g−1) | Found by this method (mean ± SD, n = 3) (ng g−1) | Average recoveries with this method (mean ± SD, n = 3) (%) | Found by LC-MS (mean ± SD, n = 3) (ng g−1) | Average recoveries in LC-MS (mean ± SD, n = 3) (%) |
---|---|---|---|---|---|
Clam | 0 | 0 | N/A | 0 | N/A |
30 | 27.74 ± 0.71 | 92.5 ± 2.4 | 26.71 ± 1.04 | 89.0 ± 3.9 | |
100 | 91.78 ± 1.17 | 91.8 ± 1.2 | 88.63 ± 3.02 | 88.6 ± 3.4 | |
200 | 188.09 ± 3.81 | 94.1 ± 1.9 | 186.75 ± 1.13 | 93.1 ± 0.6 | |
Mussel | 0 | 0 | N/A | 0 | N/A |
30 | 28.36 ± 1.08 | 94.5 ± 3.6 | 26.13 ± 0.98 | 87.1 ± 3.8 | |
100 | 91.86 ± 1.57 | 91.9 ± 1.6 | 88.85 ± 1.06 | 88.9 ± 1.2 | |
200 | 182.43 ± 2.15 | 91.2 ± 1.1 | 183.87 ± 3.39 | 91.9 ± 1.8 |
In Table 3, a comparison of this method with other HPLC-UV techniques for DA analysis reported previously is presented. Using ion-pairing/acidifying agent and reversed-phase ionic liquid dispersive liquid–liquid microextraction (RP-IL-DLLME), a highly sensitive LOD of DA was achieved, but required a time-consuming large sample volumes sample pre-concentration, with the gradient elution or the tedious LLE operation needed to be performed off line. Using SPE with SAX cartridges, MIP monolith, and magnetic MIP SPE approaches, the traditional ion-exchange interaction or MIP spatial selective recognition towards DA was adopted. Due to the non-specific nature of ion exchange and the difficulty for eluting trace target molecules from MIP materials, the detection sensitivity in these reports was in the 30–200 ng mL−1 range. In this work, massive aptamers were anchored on the MOF in the monolith for providing highly efficient bionic-affinity interactions and molecular sieving interactions, resulting in a better adsorption performance of DA, with an LOD of 7.0 ng mL−1, displaying a 5–28-fold magnitude enhancement in the detection sensitivity compared to the common SPE or MIP adsorbents reported previously. This work exhibited an online bionic recognition process of DA in shellfish with good selectivity and sensitivity, with an enhanced quantitative capacity for the analysis of DA in shellfish.
Pretreatment strategy | Absorbent | Coupled to HPLC | Extraction selectivity | LOD (ng mL−1) | Recoveries (%) | Ref. |
---|---|---|---|---|---|---|
a A large sample volume sample pre-concentration with the gradient elution.b DLLME: dispersive liquid–liquid microextraction, RP-IL: reversed-phase ionic liquid. N.: not. N.A.: Not available from the literature.c Recovery of DA from the certified mussel tissue reference material (MUS-1B). | ||||||
Ion pair | Ion-pairing/acidifying agent | Online | No | 0.04a | 97.7 ± 3.0–104.7 ± 1.5 | 36 |
DLLME | RP-ILb | Off line | No | 0.03 | N.A. | 6 |
SPE | SAX cartridges | Off line | No | 30 | 90 ± 3c | 38 |
SPME | Fe3O4@MIP | Off line | Yes | 200 | 87.6 ± 7.0–88.3 ± 6.2 | 13 |
IT-SPME | MIP monolith | Online | Yes | 76 | 89.3 ± 3.0–91.3 ± 3.4 | 23 |
IT-SPME | Aptamer@MOF monolith | Online | Yes | 7 | 91.9 ± 1.6–94.5 ± 3.6 | This work |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3ra05901d |
This journal is © The Royal Society of Chemistry 2023 |