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Self-Assembled Microgels for Sensitive and LowFouling Detection of Streptomycin in Complex Media Xiaoyan He, Huimin Han, Liqin Liu, Wenyu Shi, Xiong Lu, Jiandi Dong, Wu Yang, and Xiaoquan Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00277 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 20, 2019

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ACS Applied Materials & Interfaces

Self-Assembled Microgels for Sensitive and Low-Fouling Detection of Streptomycin in Complex Media Xiaoyan He,* Huimin Han, Liqin Liu, Wenyu Shi, Xiong Lu, Jiandi Dong, Wu Yang, Xiaoquan Lu*

Key Lab of Bioelectrochemistry and Environmental Analysis of Gansu, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070, China *Xiaoyan He: [email protected] *Xiaoquan Lu: [email protected]

Keywords: immunosensor, microgels, self-assemble, antifouling, streptomycin ABSTRACT: In terms of detection of antibiotic within complex media, the nonspecific adsorption is an enormous challenge and antifouling sensing interfaces capable of reducing the nonspecific adsorption from complex biological samples are highly desirable. In this work, a novel anti-fouling electrochemical immunosensor was explored based on the self-assemble of two kinds of poly (N-isopropylacrylamide) (P(NIPAm)) microgels on the surface of graphene oxide (GO) for sensitive detection of streptomycin (STR). The microgels modified with glycidyl methacrylate (GMA) and zwitterionic liquid 1-propyl-3-vinylimidazole sulfonate (PVIS) were prepared respectively. The microgels with GMA were used by combining of specific recognition of anti-STR. The rapidly specific binding of antigen and anti-STR

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resulted in a decrease of current density to generate electrochemical responsive signals. Zwitterionic liquid modified microgels were used as the role of anti-fouling, which can form stronger hydration and show excellent antifouling ability. As a result, we achieved efficient and sensitive detection for STR in complex sample with evidently resisted nonspecific adsorption effect, the wide linear range towards STR was from 0.05 ng mL-1 to 100 ng mL-1, with a detection limit down to 1.7 pg mL-1. The immunosensor based on the surface functionalization of microgels showed promising applications for the detection of antibiotics in complex media. 1. INTRODUCTION Streptomycin (STR) is one of the aminoglycoside antibiotics, it gives rise to mismatch of codon that achieves the purpose of sterilization and is widely used because of its effective inhibiting effect on gram-positive and gram-negative bacteria.1-3 With the wide application of STR in modern agricultural practice and foodstuffs, STR residues were frequently found in milk and other foods.4 However, excessive use of STR can cause diseases such as ototoxicity, renal toxicity and a variety of allergic reactions, which can easily do harm to people’s health.5 Many countries have set minimum levels of STR in various foods. On the grounds of the European commission, the maximum amount of STR in milk cannot exceed 200 mg/kg.6 Under these circumstances, it is urgently to develop an efficient and sensitive method to detect STR residues from foods. In recent years, various methods have been developed for the detection of STR, such as fluorescence immunoassay (FIA), 7 liquid chromatography–mass spectrometry


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(LC–MS),8 and aptasensor.9 In addition, photoelectrochemical and gas biosensors were also employed to detect antibiotics, 10,11 but the complexity of the preparation process and the high cost are their biggest drawbacks. M. H. Lan and his colleagues developed a dual-responses chemosensor for the detection of STR. 12 Although the approach exhibited fluorescent and colorimetric dual-responses to STR at submicromole level, the interference was caused because of the cross-reactions with other substances.13 Regarding the issues above, Xu and his partners designed photoelectrochemical aptasensor for the sensitive detection of STR. 14 However, the desired high sensitivity and lower detection limit are still not achieved. In conclusion, the interference from the binding of nonspecific molecules with biorecognition element is the main cause of the decreased specificity. In order to reduce interference in complex media, some reports choose to use bovine serum albumin (BSA) and 6-mercaptohexanol (MCH) based coating to block unconjugated site.2,4,9,15 liu’s group designed an electrochemical immunosensor for determination of STR in food and they used BSA to resist nonspecific adsorption. Nevertheless, the results showed that the immunosensor has highly cross reaction between other interferent and STR antibody. MCH can form hydrophilic self-assembled monolayer because it has both functional groups, -SH and -OH. However, the aptasensor for detecting STR constructed by Danesh’s group confirmed weak anti-fouling ability of MCH in complex samples. 16 Recently, many other materials were used for the construction of better anti-fouling interfaces, such as poly ethylene glycol (PEG) and its derivatives, zwitterionic polymer brushes, peptide,



oligo (ethylene glycol) (OEG) contained self-assembled monolayers (SAMs) and so on.17-22 Nonetheless, PEG and its derivatives are oxidatively damaged easily after being used for a long time and the resistance of SAMs is relatively poor in complex biological media.23 Among those materials, zwitterionic polymers with both cationic and anionic groups is a prospective candidate, 24 which can form stronger hydration via the ionic solvation and show excellent antifouling ability. 25 Hu’s group constructed zwitterionic antifouling coating via electrochemically mediated atom transfer radical polymerization (ATRP) to detect glucose. 26 Nevertheless, the method has defected mutual interference between the capabilities of immobilization and antifouling. Moreover, the ATRP process is complicated and organic halide initiator for polymerization is highly toxic. Hence, an easier immobilization and antifouling technique was imminently expected. As we know, microgel is a colloidal macromolecular network with highly porous, it shows three-dimensional structure.27,28 Microgels can be prepared simply through surfactant free emulsion polymerization, and it is possible to synthesize microgels with particular character by adding functional comonomer. 29,30 Therefore, microgels can be applied to the construction of biosensor completely. In this work, we focus on the constructing of an electrochemical immunosensor for antifouling, sensitive and detection of STR using the self-assembled functional microgels. Compared with the other methods, the approach is convenient and simple. Here, zwitterionic liquid 1-propyl-3-vinylimidazole sulfonate (PVIS) has been used to incorporate zwitterionic groups on the shell of microgels for resisting nonsepecific


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absorption. Glycidyl methacrylate (GMA) has been used to incorporate epoxy groups into microgels for further conjugation with anti-STR. Two kinds of P(NIPAm) microgels with opposite charges and abilities of antifouling and identifying were self-assembled on graphene oxide (GO) matrix. The sensor showed lower detection limit and narrower linear range to the detection of STR, specifically, high resistance toward non-specific protein molecules in complex media such as milk was proved. 2. EXPERIMENTAL SECTION 2.1. Reagents. Streptomycin polyclonal antibody (anti-STR, 1.0 mg/mL), bovine serum albumin (BSA, 98%), β-lactoglobulin (β-Lg, 90%), lipoprotein lipase (LPL, 34U/mg), streptomycin (STR, 99%) and oxytetracycline (OTC, 930U/mg) were purchased from the Shanghai Yuanye Biological Technology Co. Ltd, China. Glycidyl methacrylate (GMA), kanamycin (KAN, 94%), amoxicillin (AMX, 98%), and chloramphenicol

(CAP,

98%)

were

obtained

from

Aladdin

Reagents.

1-propyl-3-vinylimidazole sulfonate (PVIS) was synthesized by our group. 3-(methacrylamide) propyltrimethylammonium chloride (MPTC) (50% wt/wt in water) was provided by Tianjin Heowns Biochem LLC. N-isopropylacrylamide (NIPAm) was purchased from Shanghai Wujin Chemical Technology Co. Ltd. N,N'-methylenebisacrylamide (BIS) was provided by Shanghai Zhongqin Chemical Reagent Co. Ltd. Potassium peroxodisulfate (KPS) was purchased from Tianjin Kaitong Chemical Reagent Co. Ltd. Crylic acid (AA) was supplied from Tianjin Kaixin Chemical Industry Co. Ltd. Graphene oxide (GO) dispersion (5mg/mL) was purchased from Carbonium Graphite Technology Co. Ltd. (Suzhou, China). PBS



solution was prepared by disodium hydrogen phosphate dodecahydrate and sodium phosphate monobasic dehydrate. Fresh milk was purchased from a local supermarket. Other reagents were of analytical grade, and the deionized water was prepared by deionizer (Sudreli, SDLA-T-0101). 2.2. Synthesis of P(NIPAm-AA-PVIS) and P(NIPAm-MPTC-GMA) Microgels. We have prepared P(NIPAm-AA-PVIS) microgels by surfactant free emulsion polymerization.31,32 Firstly, we weighed NIPAm (0.6186 g), AA (0.0043 g), and BIS (0.0045 g) respectively, and then they were added into 50mL of deionized water. Next, oxygen was eliminated by bubbling nitrogen for 30 min with stirring. Afterwards, the solution of 0.025 g of KPS dissolved in 5.0 mL of deionized water was injected into the aboved solution to initiate the polymerization. Subsequently, AA (0.0217 g) and BIS (0.0233 g) were dissolved in 5 ml of water and slowly injected to the solution. After two or three minutes, PVIS monomer (0.015 g) which dissolved in 5.0 mL of deionized water was injected into the reaction mixture. The polymerization was performed with stirring at 70°C for 6 h. In the end, the product was dialyzed with deionized water for 4 days. And the deionized water need to be replaced about every day. The synthesis procedure of P(NIPAm-MPTC-GMA) microgels can be seen in supporting information. 2.3. Preparation of the Mixed Microgels with Anti-STR. 50 µL of anti-STR (1 mg/mL) was diluted ten times by PBS solution (pH=7.4) to 0.1 mg/mL. Initially, 500 µL of anti-STR and 500 µL of P(NIPAm-MPTC-GMA) microgels dispersion were mixed and the mixture was adequately stirred at room temperature. The above steps


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were used for completing the conjugation of anti-STR and P(NIPAm-MPTC-GMA) microgels. Then the conjugation of anti-STR and P(NIPAm-MPTC-GMA) microgels was fully mixed with P(NIPAm-AA-PVIS) microgels dispersion in a ratio of 1:1. Through the above steps, we obtained the mixed microgels with anti-STR. 2.4. Fabrication Process of the Immunosensor. Before the fabrication, the glass carbon electrode (GCE) was polished with 0.3 mm alumina slurry on the chamois. And ultrasonic washed with the mixture of deionized water and absolute ethanol, the deionized water, for 5 min in turn. Then the GCE was dried with high purity nitrogen for further modification. Subsequently, we dropped 10 µL of GO on the surface of the cleaned GCE to dried at room temperature. Next, 10 µL of the mixed microgels with anti-STR was dipped onto the surface of the GO/GCE for overnight at 4°C. The modified electrode was stored at 4°C until used it. The process of fabricating the immunosensor was monitored by the cyclic voltammetry (CV), and the parameters of CV were as below: scan ranged from -0.4 V to 0.6 V, and the scan rate was set as 0.05 V s-1. The electrochemical impedance spectroscopy (EIS) was also conducted to assess electrochemical behaviors of the immunosensor, the frequency ranged was 0.1 -10000 Hz, amplitude of the applied sine was 5 mV, and the current potential set as 0.184V. 2.5.

Nonspecific

Protein

Anti-STR/P(NIPAm-MPTC-GMA)

Adsorption

on

Microgels

and

the

Surface

of

Anti-STR/Mixed

Self-assembled Microgels. In order to evaluate antifouling ability of the immunosensor, three protein solutions (1.0 mg/mL) and real milk samples were



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detected. BSA, β-Lg and LPL were prepared into 1.0 mg/mL by PBS (10 mM, pH=7.4).21

The

anti-STR/P(NIPAm-MPTC-GMA)

microgels/GO/GCE

and

anti-STR/the mixed self-assembled microgels/GO/GCE were measured before and after incubation (incubated with different protein solution and milk for 35 min) by differential pulse voltammetry (DPV) technique in PBS (0.2 M, pH=7.4) solution containing 5.0 mM [Fe(CN)6]3-/4- and 0.1 M KCl. 2.6. Detection of STR with Immunosensor. Before every electrochemical measurement runs, 10 µL of STR standard needs to be incubated on the modified electrode for 35 minutes at room temperature. Considering that the incubation solution may evaporate, the incubated process needs to be carried out in a container. 2 The electrochemical measurement of DPV was conducted toward STR standards in the PBS (0.2 M, pH=7.4) solution containing 5.0 mM [Fe(CN) 6]3-/4- and 0.1 M KCl. The DPV peak current density was considered as the signal of the immunosensor associated with the concentration of STR. DPV measurements were from -0.1 V to 0.4 V with pulse amplitude of 50 mV and the pulse period of 50 ms. 2.7. Detection of STR in Milk. To assess the practicability of the electrochemical immunosensor in real samples, we chose milk as model. The milk sample was simply preprocessed on the basis of previously report. 33 10 µL of milk sample was incubated on the modified electrode for 35 minutes at room temperature, then the electrochemical measurement of DPV was conducted. 2.8. Instruments and Apparatus. The electrochemical workstation was used to perform all electrochemical experiments (CHI660E, Shanghai Chenhua Instrument


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Co. Ltd, China) at room temperature. A traditional three-electrode system: a GCE (diameter 5.0 mm) served as the working electrode, a saturated calomel electrode (SCE) acted as the reference electrode, and a platinum wire acted as the auxiliary electrode. The morphological measurements were performed on a scanning electron microscope (SEM, Quanta 200, philips-FEI). Fourier transformation infrared (FT-IR) spectra were recorded using a FT-IR spectrometer from Brucher, Co. Ltd, Germany. X-ray photoelectron spectroscopy (XPS) analysis was conducted with PHI-5702 multifunctional XPS. 3. RESULTS AND DISCUSSIONS 3.1. Sensing Principle of the Electrochemical Immunosensor. In this paper, an electrochemical immunosensor based on self-assembled microgel with capabilities of anti-fouling and biorecognition element immobilization as well was constructed (Scheme 1). Charged microgels were synthesized by adding ionic comonomer.32 Similarly, as a typical zwitterionic species, PVIS can be used for incorporating zwitterionic groups on the shell of microgels. And GMA is a frequently used functional monomer that has been used to incorporate epoxy groups into microgels for further conjugation with biomolecules.34 As a result, P(NIPAm-MPTC-GMA) and P(NIPAm-AA-PVIS) microgels were prepared by surfactant free emulsion polymerization.32,35

The

P(NIPAm-MPTC-GMA)

microgels

were

used

for

immobilizing anti-STR ascribing to the ring-opening reaction between epoxy groups and the amino of anti-STR.36 For the P(NIPAm-AA-PVIS) microgel, the zwitterionic group from it bound water molecules through hydrogen bonding or



strong ionic solvation,25 a tight hydration layer was formed and acted as a physical barrier to prevent the absorption of nonspecific proteins (Scheme S2). Besides, due to the respective presence of ionic comonomers AA and MPTC, the two kinds of modified microgels have opposite charges. So they were easily self-assembled on the electrode through electrostatic attraction. To improve the conductivity of the immunosensor,37 GO was dropped on the surface of GCE firstly, which could promote the transfer of heterogeneous electron.38,39 And a harmonious state would be achieved between GO and the two kinds of microgels on the electrode surface, which was attributed to the electrical properties of the three. As the target STR appeared, the antigen and antibody were specifically recognized and bound to cause signal changes. As a result, low-fouling and label-free immunosensor was simply constructed for STR detection.

Scheme 1. Schematic Illustration of the Fabrication Process for the Electrochemical

Immunosensor


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3.2.

Characterization

of

Synthesized

Microgels.

The

formation

of

P(NIPAm-MPTC-GMA) and P(NIPAm-AA-PVIS) microgels were investigated by FTIR spectroscopy. FTIR absorption spectra of P(NIPAm-AA-PVIS) microgel and other related monomers were investigated (Figure S1). Some changes from the absorption peaks of P(NIPAm-AA-PVIS) can be observed compared to the related monomers. The absorption peaks at 1627 cm-1 and 1620 cm-1 were responsible for the disappearance of C=C in AA and NIPAm because of polymerization. 40 Additionally, the presence of absorption peak at 1176 cm-1 was corresponding to S=O in SO3 - of PVIS and the band at 1647 cm-1 stemming from stretching vibrations on the imidazole chain band showed a slight shift.41 The absorption peak at 3515 cm-1 was due to the stretching vibrations of O-H in COOH of AA. The FTIR absorption spectra of P(NIPAm-MPTC-GMA) microgels and other related monomers were also illustrated (Figure S2). The presence of absorption band at 920 cm-1 was responsible for epoxy group of GMA. The peak at 1653 cm-1 corresponded to the stretching vibration of C=C characteristic absorption of MPTC and the peak at 1633 cm-1 for C=C of GMA disappeared as a results of polymerization. In summary, all of the FTIR spectral bands proved the P(NIPAm-MPTC-GMA) and P(NIPAm-AA-PVIS) microgels were synthesized successfully. XPS analysis was used to confirm the modification of GO and the mixed microgels with anti-STR. As shown in Figure 1, C 1S (287 eV), N 1S (403 eV), and O 1S (536 eV) were observed after the glassy carbon electrode was modified with GO (Figure 1A). From the Figure 1B, the peak of C 1s and O 1S decreased relatively after



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modified the mixed self-assembled microgels with anti-STR, but the peak of N 1S evidently increased. Meanwhile, the peak at 195 eV, corresponding to the Cl 2p from P(NIPAm-MPTC-GMA) microgels turn up. The peaks at 160 eV and 134 eV, corresponding to the S 2p and P 2p respectively appeared. It showed that the P(NIPAm-AA-PVIS) microgels and the anti-STR were successfully modified on the surface of the GO/GCE.

Figure 1. XPS spectra of (A) GO/GCE and (B) anti-STR/the mixed self-assembled microgels/GO/GCE. The morphology of the electrodes modified with different materials were monitored with the SEM. From the Figure 2a, we can observe that the GO was uniformly spread on the surface of the bare GCE, and there was a little fold. After modified with the mixed P(NIPAm) microgels, two kinds of P(NIPAm) microgels with opposite charges were evenly arranged on the surface of the GO/GCE (Figure 2b). The phenomenon is attributed

to

the

electrostatic

attraction

between

negatively

charged

P(NIPAm-AA-PVIS) microgels and positively charged P(NIPAm-MPTC-GMA) microgels. Another reason is ascribed to the negatively charged GO, the charged three elements maintained a balance, which helps to form the evenly distributed and


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self-assembled construction.

Figure 2. SEM images of (a) GO-modified GCE and (b) the mixed self-assembled microgels/GO/GCE. 3.3. The Antifouling Ability of the Immunosensor. To assess the antifouling behavior of the immunosensor interface based on the self-assembled microgels, three single protein solutions (BSA, β-Lg and LPL were prepared into 1.0 mg/mL by PBS (10mM, pH=7.4)) and unprocessed real milk sample were detected by DPV method. In comparison, the anti-STR/P(NIPAm-MPTC-GMA)/GO-modified electrode without P(NIPAm-AA-PVIS) microgels was also prepared by electrostatic attraction between GO

and

P(NIPAm-MPTC-GMA)

microgels.

The

DPV responses

of the

immunosensor with P(NIPAm-AA-PVIS) microgels and the immunosensor without P(NIPAm-AA-PVIS) microgels were compared before and after incubation (incubated with single protein solution and milk for 35 min). As shown in Figure 3A and Figure 4A, the changes of DPV response of the immunosensor without P(NIPAm-AA-PVIS) microgels were significantly after incubation in protein solutions.

The

DPV

current

response

of

the

anti-STR/P(NIPAm-MPTC-GMA)/GO-modified electrode changed 85%, 89% and



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69% of the original value after incubated BSA, β-Lg and LPL respectively. These were attributed to the fact that proteins adsorbed onto the surface of the electrode and the original electronic transmission process was changed after incubation, giving rise to obvious changes in the DPV signals. 42 On the contrary, the very tiny changes of DPV signals of the immunosensor with P(NIPAm-AA-PVIS) microgels were observed after incubation in BSA, β-Lg and LPL solutions apart (Figure 3B). The DPV current response change to BSA was 8.5%, and the changes to β-Lg and LPL were 2.7% and 0.5% respectively. It can be seen that the P(NIPAm-AA-PVIS) microgels can prominently reduce the proteins adsorption, which suggested promising antifouling ability of the immunosensor with P(NIPAm-AA-PVIS) microgels. In addition, as shown in Figure 4B, after incubation in different unprocessed milk samples,

the

DPV

peak

current

density

of

the

immunosensor

without

P(NIPAm-AA-PVIS) microgels evidently changed. However, the immunosensor with P(NIPAm-AA-PVIS) microgels had only a little impact on the DPV signal. The above results were attributed to the fact that the P(NIPAm-AA-PVIS) microgels have outstanding ability to resist nonspecific protein adsorption owing to the formation of stronger hydration via the ionic solvation from the zwitterionic polymer. Therefore, the immunosensor based on the self-assembly microgels was proved to powerfully resist different nonspecific protein adsorption in the complex media.


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Figure

3.

DPV

responses

of

the

anti-STR/P(NIPAm-MPTC-GMA)

microgels/GO/GCE (A) and the anti-STR/ mixed self-assembled microgels /GO/GCE (B) before (red line) and after (black line) incubation in three protein solutions (1: BSA; 2: β-Lg; 3: LPL, 1.0 mg mL-1 each).



Figure 4. DPV peak current density [Δip/ip0(%)] changes of immunnosensor with P(NIPAm-AA-PVIS) microgels or without P(NIPAm-AA-PVIS) microgels after incubated in different proteins (A) and unprocessed real milk samples (B). 3.4. Electrochemical Behaviors of the Immunosensor. The immunosensor was successfully prepared through a two-step fabrication process. Firstly, the GCE was modified with GO through drip method. Next, two kinds of microgels with anti-STR was dropped on the GO/GCE and self-assembled. The electrochemical behaviors of the immunosensor was monitored using EIS and CV, as shown in Figure 5. According to the characterization of CV, the GO-modifed electrode (Figure 5A, curve b) showed much lower peak current density compared with the bare GCE (Figure 5A, curve a). The phenomenon be ascribed to the electrolyte of PBS (0.2 M, pH=7.4) solution containing 5.0 mM [Fe(CN)6]3-/4- and 0.1 M KCl. The negatively charged GO and the [Fe(CN)6]3-/4- solution were mutually exclusive, which led to the impediment of interfacial electron transfer.38 However, we could evidently see the peak current density was enhanced after modified the mixed self-assembled microgels with anti-STR (Figure 5A, curve c). The consequence may result from the following


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reasons. As we know, the mixed self-assembled microgels with anti-STR was consist of positively charged P(NIPAm-MPTC-GMA) microgels, negatively charged P(NIPAm-AA-PVIS) microgels and anti-STR. And anti-STR may be positively charged in the environment of whole system because of the isoelectric point of anti-STR was more than pH=7.4. It led to the mixed self-assembled microgels with anti-STR generally exhibited electropositivity. So the mixed self-assembled microgels with anti-STR has electrostatic attraction to [Fe(CN) 6]3-/4-, additionally, GO can promote electron transfer on the sensing interface. As a result, the peak current density of anti-STR/the mixed self-assembled microgels/GO/GCE was much higher than the GO-modified GCE. EIS characterizations of different modified electrodes were illustrated in Figure 5B. We all know that the semicircle part at higher frequency range represents the resistance of charge transfer (Rct) in the Nyquist impedance plot. As shown in Figure 5B, the Rct of GO/GCE (3097 Ω, cure b) significantly higher than other modified electrodes attributed to the function of mutual repulsion between GO and [Fe(CN)6]3-/4-. Nevertheless, the Rct decreased after modified the mixed self-assembled microgels with anti-STR, which due to the electrostatic attraction between electropositive mixed self-assembled microgels with anti-STR and electronegative [Fe(CN)6]3-/4- probe (339.9 Ω, curve c). While incubated in 10 ng mL-1 of STR standard for 35 minutes, the Rct of the modified electrode increased again (521.6 Ω, curve d). It was ascribed to the binding of antigen and anti-STR. The binding gave rise to the retarding of the interfacial electron transfer. Meanwhile, from the CV characterization, after incubated in 10 ng mL-1 of STR standard solution, the



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peak current value was less than that without STR (Figure 5A, curve d). The corresponding electrochemical parameters from CV and EIS were listed in Table 1.

A

B

Figure 5. CV curves (A) and EIS (B). (a) GCE; (b) GO-modified GCE; (c) anti-STR/the mixed self-assembled microgels/GO/GCE; (d) STR/anti-STR/the mixed self-assembled microgels/GO/GCE. Table 1. Electrochemical Parameters of Different Modified Electrodes Extracted from CV and EIS Electrode

Ipa(μA)

Ipc(μA)

Rs (Ω)

Rct (Ω)

GCE

48.05

-34.73

6.631

261.1

GO/GCE

4.579

-3.505

16.22

3097

anti-STR/the mixed self-assembled microgels/GO/GCE

12.71

-9.916

10.34

339.9

STR/anti-STR/the mixed self-assembled microgels/GO/GCE

11.38

-6.111

10.71

521.6

3.5. Optimization Trials. In order to get an optimal sensing condition, the ratio of the two kinds of microgels, pH and the incubation time for antigen-antibody interaction need to be optimized. Firstly, we mixed the two kinds of microgels in four different ratios, P(NIPAm-AA-PVIS) microgel accounted for 50%, 100%, 150% and


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200% of the volume of P(NIPAm-MPTC-GMA) microgel, respectively. Then we chose β-Lg as a nonspecific protein to explore the optimal microgel ratio. As seen from

Figure

6a,

when P(NIPAm-AA-PVIS)

accounts

for

100%

of the

P(NIPAm-MPTC-GMA) volume, the change in DPV peak current was minimal. The result indicated that anti-fouling and specific recognition reached a balance at the ratio of the two kinds of microgels was 1:1. DPV characterization of the developed immunosensor was also performed in different pH of PBS (0.2 M) solution containing 5.0 mM [Fe(CN)6]3-/4- and 0.1 M KCl to optimize the pH of the electrolyte. As seen in Figure 6b, the current density was the biggest at pH 7.4. Therefore, 7.4 was the most favorable pH value for electrochemical sensing. The Figure 6c indicated that DPV peak current density gradually increased with the incubation time, and remained stable over 35 min. This result showed that the binding of antigen and antibody reached equilibrium at 35 min, as a result, we chose 35 minutes as the optimal incubation time.

a

b

c

Figure 6. The impact of (a) the ratio of the two kinds of microgels, (b) pH of the solution, and (c) incubation time on the electrochemical response of the immunosensor.



3.6. Analytical Performance of the Immunosensor. The performance of the developed immunosensor for the determination of STR was studied in PBS (0.2M, pH=7.4) by DPV. As we seen in Figure 7b, the DPV response change (I(μA)) behaved as a good linear correlation (R2=0.997) with logarithm values of the STR standard concentrations in the range of 0.05 ng mL-1 to 100 ng mL-1. The regression equation was I(μA) = -15.61+1.7logC[STR] (ng mL-1), and the limit of detection (LOD) was approximately 1.7 pg mL-1 (S/N = 3). The LOD of this designed immunosensor is dramatically lower than other reports for the detection of STR. 14,43,44 The low detection limit was mainly ascribed to the sensing interface based on the self-assembly multifunctional microgels. To clarify the results, as shown in Table 2, the performance were compared with the previous techniques. It showed that the anti-STR binding efficacy and antifouling ability of the zwitterionic microgels were outstanding in this paper.

Figure 7. (a) DPV curves of the electrochemical immunoassay toward STR standards in PBS solution (0.2 M, pH= 7.4 ); (b) Linear fitting curve between the logarithm values of STR concentrations and DPV peak current density.


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Table 2. Comparison of the Designed Immunosensor with Other Methods for STR Detection method

linear range

Limit of detection

references

0.05-50 ng mL-1

5 pg mL-1

2

0.1-100 nM

36.45 pM

9

2–70 μM

0.2 μM

12

0.1-100 nM

48.1 pM

44

0.1-50 nM

33 pM

45

6×10-2-2.0 μM

54.5 nM

46

0.3-50 ng mL-1

0.3 ng mL-1

47

0.05-100 ng mL-1

1.7 pg mL-1

-

Electrochemical immunosensors

Electrochemical aptasensor Fluorescent and colorimetric dual-responses chemosensor Photoelectrochemical aptasensor Photoelectrochemical aptasensor Fluorescent aptasensor FIA-EQCN biosensor

This work

To test the reproducibility and repeatability of the immunosensor, five modified electrodes were prepared at the same time for the detection of STR (20 ng/mL), it was calculated that the RSD was 3.80%. Then the same one immunosensor was used to detect 20 ng/mL of STR repeatedly for six times, the result showed that the RSD was 3.13%. Therefore, the reproducibility and repeatability of this sensor were satisfactory. The selectivity of the developed immunosensor was also evaluated. We selected four different interferents containing OTC, KAN, AMX, CAP to perform the task for several times. Furthermore, the concentrations of these interferents were one hundred times of the target STR. As illustrated in Figure 8, the change of DPV peak density (Δip/ip0(%)) of the designed immunosensor was most significant toward STR. In



contrast, the responses for OTC, KAN, AMX and CAP were so tiny that they can be neglected. In consequence, the above interference experiment fully demonstrated the excellent specificity of the immunosensor.

Figure 8. Responses of the immunosensor to 2μg/mL CAP, 2μg/mL KAN, 2μg/mL AMX, 2μg/mL OTC, and 20ng/mL STR. The error bars represent the standard deviation of three repeated measurements. To verify the stability of the constructed electrochemical immunosensor, the CV was used for exploring. As shown in Figure S4, 50 cycles of CV measurement was performed in the PBS (0.2M, pH=7.4) solution containing 5.0 mM [Fe(CN) 6]3-/4- and 0.1 M KCl. We acquired that the peak current density value can still reach 92% of the initial value after 50 cycles. In addition, the DPV peak current of the immunosensor was only decreased by 7.4% after the electrode was stored at 4℃ for 10 days. It showed that the immunosensor had favorable stability. 3.7. Analysis of STR in Milk. The electrochemical immunosensor was used for the detection of STR residue in real milk sample. After the milk sample was removed the fat precipitation by simply centrifugate, the STR standard of different concentration


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were spiked into milk sample. The linear detection ranges of STR concentration in milk were from 0.05 ng mL-1 to 100 ng mL-1 . And the analytical results were summarized in Table 3 with a high recovery. The results showed that the immunosensor we constructed was also highly practical for complex real samples. Table 3. Analytical Results for STR in Real Milk Samples

samples

Added STR （ng mL-1）

Detected （ng mL-1）

Recovery (%)

RSD (%, n=3)

1 2

0.5 15

0.4926 15.34

98.1 102.1

1.2 1.9

3

50

51.95

103.9

1.2

4. CONCLUSION In summary, we developed a novel immunosensor for ultrasensitive, low-fouling detection of STR by means of the self-assembling of two kinds of opposite charged P(NIPAm) microgels. The method is simple and convenient to construct the immunosensor because self-assembling characteristic of charged functional materials were used. The immunosensor both have two capabilities of anti-fouling and biorecognition element immobilization, which provided an excellent approach for detecting STR, even though within the complex milk media. As a result, the wide linear range towards STR was from 0.05 ng mL-1 to 100 ng mL-1, the LOD for STR was measured as low as 1.7 pg mL-1. The electrochemical immunosensor is significantly potential to apply in complex media for the sensitive and low-fouling detection of other biomolecules.



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ASSOCIATED CONTENT Supporting Information. The

scheme

of

the

electrochemical

workstation,

synthesis

of

P(NIPAm-MPTC-GMA) microgels, the scheme of synthesis and antifouling of P(NIPAm-AA-PVIS) microgels, FTIR of NIPAm, AA, PVIS, MPTC, GMA, P(NIPAm-MPTC-GMA) and P(NIPAm-AA-PVIS) microgels, the charge transport characteristic and stability of the anti-STR/mixed self-assembled microgels/GO/GCE. AUTHOR INFORMATION Corresponding Author *Xiaoyan He: [email protected] *Xiaoquan Lu: [email protected] Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by the National Natural Science Foundation of China (NSFC) (No. 21765019, No. 21665024). REFERENCES (1) Baxter, G. A.; Ferguson, J. P.; O'Conno, M. C.; Elliott, C. T. Detection of Streptomycin Residues in Whole Milk Using an Optical Immunobiosensor. J. Agric.


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