A Biosensor Combining Molecularly Imprinted Polymers (M-MIPs) and Surface Enhanced Raman Spectroscopy (SERS) to Detect Antibiotics in Food Samples
Yi Sun,1*, Jon Ashley1, Kaiyu Wu1, and Anja Bosen1.
1 Department of Micro- and Nanotechnology, Technical University of Denmark, Ørsteds Plads, DK-2800 Kgs. Lyngby, Denmark
* Email: [email protected]; Tel.: +45 45256319
In this study, temperature-responsive magnetic molecularly imprinted polymers (M-MIP) nanoparticles were synthesized for the first time for the extraction of cloxacillin in pork products. By combining the M-MIPs with surface enhanced Raman spectroscopy (SERS), a sensitive biosensor was demonstrated to detect cloxacillian with pico-mole sensitivity.
MIPs are synthetic ligands which can be tailored to bind any analyte of choice1. They are of great interest due to their thermal stability, robustness, low cost and comparable binding affinity. They have been used in sample preparation and biosensing as an attractive alternative to natural antibodies to capture targets ranging from small molecules to big proteins.
In this work, the magnetic nanoparticles with MIP-based receptors were synthesized for efficient and rapid extraction of antibiotic residues in pork samples. Fe3O4 nanoparticles were obtained using the solvothermal synthesis. The resultant nanoparticles were treated with Tetraethyl orthosilicate (TEOS) to form a SiO2 layer. Finally a thin MIP layer was polymerized round the nanoparticles using azobisisobutyronitrile (AIBN) as the initiator, ethylene glycol dimethacrylate (EDGMA) as the cross-linker, N-isopropylmethacryamide (NIPAm), methacrylic acid (MAA) as the monomers and the antibiotic as the template. By adding the monomer NIPAm, the MIPs become temperature responsive, and can swell at low temperature to release the target. The corresponding magnetic non-imprinted polymer nanoparticles (M-NIP) was prepared using the same method in the absence of the template. An Overview of the synthesis strategy is shown in Fig. 1. The resultant M-MIP nanoparticles were characterized using IR, XRD scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (Fig. 2). Both binding affinities of the resultant M-MIPs and M-NIPs were tested using UV absorbance (Fig. 3). M-MIPs with 300-400 nm in size and good binding capacities were obtained.
To demonstrate the feasibility of using M-MIPs for sample preparation, the synthesized M-MIPs were mixed with pork blood samples spiked with Chloxacillian. After incubation at room temperature, the M-MIPs were collected using a magnet and washed by acetonitrile. Owing to the thermos-responsive properties of MIPs, Chloxacillian was easily released by cooling the MIPs to 4 degree. The collected Chloxacillian was dropped on a SERS substrate which contained an array of silicon micropillars coated with silver. The corresponding calibration plots showed a detection limit (LOD) of about 50 pmol (Fig. 4). The biosensor combining M-MIPs and SERS would be widely used on site or in the field for rapidly screening food contaminants to ensure food safety.
Fig. 1: Overview of the synthesis of M-MIPs
Fig.2 IR characterization of Fe3O4, Fe3O4@SiO2, Fe3O4@ SiO2-MPA and, Fe3O4@SiO2-MIP; XRD of Fe3O4.
Fig.3 (A) Binding kinetics and (B) Binding capacity of Cloxacillian MIPs and NIPs.
Fig.4 SERS spectra of cloxacillin in MeOH:acetic acid (9:1) and corresponding calibration plots.
REFERENCES:
- J. Ashley, M-A. Shahbazi, K. Kant, V. A. Chidambara, A.Wolff, D. D. Bang, Y. Sun, “Molecularly Imprinted Polymers for Sample Preparation and Biosensing in Food analysis: Progress and Perspectives, Biosens. Bioelectron. 2017, 91, 606-615.
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Progress Claims
This article presents a novel method to bond PLA, a 3D-printing material, and PMMA, a popular substrate material for microfluidic applications. With this technique, the tubing connectors can be fabricated by 3D printing and make this PLA/PMMA hybrid microfluidic chip extremely easy to use for experiments. The major challenge of bonding this hybrid chip is the high level surface roughness of PLA substrates with its significant influences on bonding strength [1]. After Ethanol treatment and UV irradiation of this hybrid chip, a post-annealing step was realized to facilitate the bonding. To further analyze the bonding quality leakage test, cross-sectional image by microscope, and pressure bursting test were conducted. The experiment results clearly showed that this method could successfully and rapidly form a strong bond (>13 bars) between PLA and PMMA substrates.
Background
Nowadays, thermoplastics are used commonly in microfluidic applications. The bonding of PMMA/PLA can offer significant benefits taken advantages from 3D-printing process such as allowing producing incredibly complex products in a short time, while minimizing material waste.
Description of Bonding Procedure
Ethanol solution was distributed uniformly sandwiched between two substrates by spin-coating process (190 rpm, 10 sec). Following the UV irradiation (56 sec), an instantaneous and permanent bonding can be formed between PMMA and PLA. However, the bonding strength is significantly affected by the high level of surface roughness of PLA substrates (range from 4.5 to 6.5μm). It can cause the failure of bonding. To solve this critical issue, we employed a post-annealing strategy (55℃, 30 min) straightforwardly after UV exposure step. Its purpose is help create more contacting points between two bonded substrates because of surface degradation caused by coarsening phenomena [3]. Besides post annealing can help relieve stress and therefore improve the bonding strength. The temperature executed for post-annealing is below the glass transition temperature of PLA (Tg of PLA = 60~65℃), hence there has no significant channel deformation observed. The overall bonding procedure is described in Fig. 1.
After bonding, several experiments were conducted to characterize the bonding quality such as leakage tests, cross-sectional images by microscope, and burst tests.
Experimental Results
- Comprehensive examination
Following the completion of the bonding experiments, leakage tests and the cross sectional investigation using microscope of bonded chip were conducted, the results of which are presented in Fig. 2. The figure clearly demonstrates the effectiveness of the proposed bonding method for heterogeneous substrates between PMMA/PLA. Figure 2(a) shows the bonded chip, Figure 2(b) shows the enlarged figure of the microchannel, both figures clearly showed that no leakage was observed. Figure 2(c) shows the cross-sectional image of bonded chip.
- The influence of post-annealing conditions on bonding strength.
Fig.3 illustrates the set up for busting test and Table 1 lists the experiment results of bonding strength. In all of the experiments, the microchannels were fabricated on the PMMA substrates and the cover substrates were PLA. Table 1 clearly shows that the microfluidic chips have sufficient bonding strength above 10 bars.
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