Single-Template Molecularly Imprinted Chiral Sensor for

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Single-Template Molecularly Imprinted Chiral Sensor for Simultaneous Recognition of Alanine and Tyrosine Enantiomers Qianqian Zhao, Jiapei Yang, Jie Zhang, Datong Wu, Yongxin Tao, and Yong Kong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b03426 • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 3, 2019

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Analytical Chemistry

Single-Template Molecularly Imprinted Chiral Sensor for Simultaneous

Recognition

of

Alanine

and

Tyrosine

Enantiomers

Qianqian Zhao, Jiapei Yang, Jie Zhang, Datong Wu, Yongxin Tao, and Yong Kong*

Jiangsu Key Laboratory of Advanced Catalytic Materials and Technology, Changzhou University, Changzhou 213164, China

Email: [email protected] Tel.: 86-519-86330253. Fax: 86-519-86330167. 1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Chiral recognition of L-amino acids is of significant importance due to the crucial role of L-amino acids in life sciences and pharmaceutics. In this work, a chiral sensor with capability of probing two chiral amino acids by an attractive single-template molecular imprinting strategy is introduced and used in the simultaneous chiral recognition of D/L-alanine (D/L-Ala) and D/L-tyrosine (D/L-Tyr). The assay relies on the hydrolysis of L-alanyl-L-tyrosine dipeptide doped in silica/polypyrrole (SiO2/PPy) under acidic conditions, resulting in L-Ala and L-Tyr co-imprinted chiral sensor. This work opens up a new avenue for simultaneous chiral sensing of two or more chiral amino acids by incorporating only one template, circumventing the shortcomings encountered with multi-template molecularly imprinted technology.

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Chirality (handedness) is an intrinsic feature of most biological molecules, and the determination of enantiomeric purity has been considered as a challenging task for medical and food industry.1‒4 As the crucial components in biological system, amino acids and their derivatives are chiral except for glycine, and only one enantiomer (L-amino acids) is of interest and exhibits biological activity.5,6 Therefore, the enantioselective determination of chiral amino acids is of significant importance in the fields of life sciences and pharmaceutics. The mechanisms of molecular imprinting,7‒9 ligand exchanging10‒12 and supramolecular interactions13‒16 are widely accepted in the enantioselective determination of chiral amino acids in the past decades. Especially, molecular imprinting is an attractive approach of creating artificial recognition sites which are complementary to the template (or imprint) molecules in their shape, size and spatial arrangement of the chemical functionalities.17,18 Usually, molecularly imprinted materials can be categorized into two types: single-template imprinted materials19,20 and multi-template imprinted materials.21,22 The former aims to create sites for one kind of target molecules, while the latter is designed for simultaneous selective recognition of two or more kinds of targets. Because the impartation of multiple recognition sites in one solid material is advantageous to sense more than one compound at a time and greatly extend the application of molecular imprinting technique (MIT), it has attracted growing interest since the first proposal of this technique in 1999.23 In that proposal, Sreenivasan and Sivakumar reported the preparation of poly(2-hydroxy ethyl methacrylate) simultaneously imprinted with salicylic acid and hydrocortisone via gamma irradiation, which indicates the possibility by imprinting sites for two compounds. Traditional multi-template imprinted materials are prepared by the polymerization of monomers in the 3 ACS Paragon Plus Environment


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presence of multi-template molecules, followed by removal of the multiple templates and formation of complementary cavities matched well with the templates. However, it usually encounters negative situations such as uneven distribution of the multiple templates in the matrix, competitive doping of the multiple templates in the matrix and the consequent uncontrollable doping amount of the multiple templates. Here, we first report on the fabrication of a single-template molecularly imprinted chiral sensor for simultaneous recognition of alanine (Ala) and tyrosine (Tyr) enantiomers. Polymer materials are widely used as the matrix in traditional MIT, however, deformation or even destruction of the imprinted cavities in polymer matrixes often occurs during the templates removal by polar organic solvents due to the low mechanical strength of polymers. As previously reported, the presence of inorganic oxides such as SiO224 and MnO225 in the matrixes of MIT can significantly enhance the mechanical strength of the matrixes, and therefore

silica/polypyrrole

(SiO2/PPy)

is

used

as

the

matrix

in

this

work.

L-Alanyl-L-tyrosine dipeptide is first doped into the matrix, which is then hydrolyzed under acidic conditions to produce L-alanyl (L-Ala) and L-tyrosine (L-Tyr). After the removal of L-Ala and L-Tyr, L-Ala and L-Tyr co-imprinted SiO2/PPy is obtained, which is fully characterized by Fourier transform infrared (FT-IR) spectra, circular dichroism (CD) spectra, X-ray diffraction (XRD), SEM, TEM and electrochemical impedance spectroscopy (EIS). Finally, a glassy carbon electrode (GCE) modified with the L-Ala and L-Tyr co-imprinted SiO2/PPy is used as the electrochemical chiral sensor for simultaneous recognition of D/L-Ala and D/L-Tyr, and the parameters are also optimized to achieve the highest recognition efficiency. 4 ACS Paragon Plus Environment

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EXPERIMENTAL SECTION Reagents and Apparatus. D/L-Ala, D/L-Tyr, pyrrole and tetraethoxysilane (TEOS) were purchased from Aladdin Chemistry Co., Ltd (Shanghai, China). Ferric chloride hexahydrate (FeCl3·6H2O), ammonia (NH3·H2O), potassium ferrocyanide (K4Fe(CN)6), potassium ferricyanide (K3Fe(CN)6) and concentrated hydrochloric acid (HCl) were received from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). L-Alanyl-L-tyrosine was purchased from Hefei Guotai biotechnology Co., Ltd (Hefei, China). All aqueous solutions were prepared with ultrapure water (Milli-Q, Millipore). Pyrrole was distilled under reduced pressure before use. The FT-IR and the CD spectra of different samples were recorded by means of a Nicolet FTIR-8400S spectrometer (Shimadzu, Japan) and a CD J-1500 spectrometer (JASCO, Japan), respectively. The XRD patterns of different samples were measured by using a D/max 2500PC diffractometer (Rigaku, Japan). The morphologies of dipeptide doped SiO2/PPy and L-Ala and L-Tyr co-imprinted SiO2/PPy were recorded with a Supra55 field emission scanning electron microscope (FESEM, Zeiss, Germany) and a JEM 2100 transmission electron microscope (TEM, JEOL, Japan), respectively. Electrochemical measurements were carried out in a traditional three-electrode cell connected to a CHI-660D electrochemical workstation. A GCE modified with the L-Ala and L-Tyr co-imprinted SiO2/PPy was used as the working electrode, and a platinum plate (10  5 mm) and a KCl saturated Ag/AgCl electrode were taken as the counter electrode and the reference electrode, respectively. Preparation of Dipeptide Doped SiO2/PPy. First, SiO2 nanospheres were prepared according to the method previously reported with slight modification.26 Briefly, 1.8 mL of 5 ACS Paragon Plus Environment


TEOS was added into the mixture of 24.7 mL alcohol, 9.9 mL H2O and 3.6 mL NH3·H2O, and the solution was agitated by a magnetic stirrer for 2 h. The products were centrifugally separated, washed and freeze-dried at –49 °C for 6 h, and then the obtained SiO2 nanospheres were dispersed in 10 mL water as a 20 wt% solution under ultrasonic vibration. Next, 45 μL of pyrrole (0.65 mmol) and 15 mg of L-alanyl-L-tyrosine were added into the SiO2 dispersion and stirred vigorously for 24 h before being cooled to 0 °C, and then 10 mL of FeCl3 solution (1.52 M) was added dropwise to the above mixture. After continuous stirring for 6 h, the resultant L-alanyl-L-tyrosine doped SiO2/PPy was centrifugally separated, washed and freeze-dried at –49 °C for 3 h. Preparation of L-Ala and L-Tyr Co-Imprinted SiO2/PPy. The L-alanyl-L-tyrosine doped SiO2/PPy was added into 10 mL of HCl aqueous solution (3 M), and then the doped dipeptide was hydrolyzed at 130 °C for 2 h.27 The hydrolysates, L-Ala and L-Tyr, were removed by the mixture of alcohol and acetic acid (v/v, 10:1), and the resultant L-Ala and L-Tyr co-imprinted SiO2/PPy was dried at 60 °C for 4 h. For control experiments, non-imprinted SiO2/PPy (NI-SiO2/PPy) was also prepared by the same procedure except for the addition of the L-alanyl-L-tyrosine dipeptide. Simultaneous Chiral Recognition of D/L-Ala and D/L-Tyr. First, a chiral sensor based on the L-Ala and L-Tyr co-imprinted SiO2/PPy was fabricated. In a typical procedure, 2 mg of the co-imprinted SiO2/PPy was dispersed in 1 mL of ultrapure water, and then 10 μL of the dispersion was cast onto the surface of a pre-cleaned GCE (3 mm in diameter) and allowed to dry at room temperature (25 °C). For control experiments, NI-SiO2/PPy modified GCE was also prepared by the same procedure. 6 ACS Paragon Plus Environment

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Next, simultaneous chiral recognition of D/L-Ala and D/L-Tyr with the as-fabricated chiral sensor was studied by differential pulse voltammetry. The chiral sensor was immersed into 25 mL of 0.1 M phosphate buffer solution (PBS, pH = 7.0) containing L-amino acids (2 mM L-Ala and 1 mM L-Tyr) or D-amino acids (2 mM D-Ala and 1 mM D-Tyr), and then a positive potential of +0.3 V was applied at the chiral sensor for 1000 s for the rebinding of Land D-amino acids. After that, the differential pulse voltammograms (DPVs) of L- and D-amino acids rebound to the chiral sensor were recorded at 25 °C to assess the recognition ability of the chiral sensor. In addition, the DPVs of L- and D-amino acids rebound to the NI-SiO2/PPy modified GCE were also recorded for a comparison. Figure 1 is the schematic illustration showing the basic strategy for the fabrication of the L-Ala and L-Tyr co-imprinted SiO2/PPy and simultaneous chiral recognition of D/L-Ala and D/L-Tyr.

Figure 1. Schematic illustration showing the basic strategy for the fabrication of L-Ala and L-Tyr co-imprinted SiO2/PPy and simultaneous chiral recognition of D/L-Ala and D/L-Tyr. RESULTS AND DISCUSSION FT-IR Spectra of L-Ala and L-Tyr Co-Imprinted SiO2/PPy. Figure 2 shows the FT-IR spectra of dipeptide, dipeptide doped SiO2/PPy before and after hydrolysis, and L-Ala and 7 ACS Paragon Plus Environment


L-Tyr co-imprinted SiO2/PPy. For the dipeptide doped SiO2/PPy (curve a), the characteristic bands centered at 1080 and 925 cm–1 are related to the O–Si–O asymmetric stretching, and the vibrations of Si–OH bond can be visualized in the region of 792 cm–1.28 Another two bands located at 1550 and 1458 cm–1 can be attributed to the C–C and C–N stretching vibrations in pyrrole ring, respectively.29 The band at around 1676 cm–1 is assigned to the stretching vibrations of C=O from –NHCO– in the dipeptide doped in SiO2/PPy, which is also observed on the spectrum of L-alanyl-L-tyrosine dipeptide (curve b). Interestingly, the characteristic band of dipeptide (1676 cm–1) disappears completely after the hydrolysis in HCl solution, meanwhile, several new bands can be observed at 3084, 2111, 1620 and 1515 cm–1, respectively (curve c). The bands at 3084, 1620 and 1515 cm–1 are indicative of the presence of amino acids containing NH3+ group,30 and another band at 2111 cm–1 is due to the combination of NH3+ deformation and NH3+ torsion.31 Thus it is confirmed that the L-alanyl-L-tyrosine dipeptide is completely hydrolyzed to L-Ala and L-Tyr and the amino groups of L-Ala and L-Tyr are protonated to NH3+ in HCl solution. After the removal of L-Ala and L-Tyr by the mixture of alcohol and acetic acid, it is noteworthy that these characteristic bands of NH3+ group disappear on the spectrum of the co-imprinted SiO2/PPy (curve d), suggesting the successful removal of L-Ala and L-Tyr and the formation of L-Ala and L-Tyr co-imprinted SiO2/PPy.


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a

Transmittance / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1676 1458 1550

b

792 925 1080

c

1676 2111

d

925 792 1620 1515

3084

1080 15501458

925

792

1080

4000

3500

3000

2500

2000

1500

-1

1000

500

Wavenumber / cm

Figure 2. FT-IR spectra of dipeptide doped SiO2/PPy (a), L-alanyl-L-tyrosine dipeptide (b), dipeptide doped SiO2/PPy after hydrolysis (c), and L-Ala and L-Tyr co-imprinted SiO2/PPy (d). CD Spectra of L-Ala and L-Tyr Co-Imprinted SiO2/PPy. Figure 3 shows the CD spectra of dipeptide doped SiO2/PPy before and after hydrolysis, and L-Ala and L-Tyr co-imprinted SiO2/PPy. For L-alanyl-L-tyrosine doped SiO2/PPy (curve a ), the negative CD signal at around 175 nm and the positive CD signal at around 220 nm are indicative of the secondary structure of peptides and proteins,32 suggesting successful doping of L-alanyl-L-tyrosine in SiO2/PPy. After the hydrolysis in HCl solution, the two CD signals disappear completely, indicating the decomposition of the L-alanyl-L-tyrosine dipeptide; meanwhile, three new positive CD signals are observed (curve b). Among the three new positive signals, the signal centered at around 190 nm is assigned to L-Ala,32 and another two signals centered at around 250 and 280 nm can be attributed to L-Tyr.33 The appearance of such new CD signals clearly indicates the production of L-Ala and L-Tyr through the hydrolysis of the L-alanyl-L-tyrosine dipeptide. Noted that there is no signal on the CD spectrum of the co-imprinted SiO2/PPy (curve c), demonstrating that both L-Ala and L-Tyr 9 ACS Paragon Plus Environment


are completely removed after being washed with the mixture of alcohol and acetic acid. 20 15 10

CD / mdeg

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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5 c

0

a

-5

b

-10 -15 150

200

250

300

Wavelength / nm

Figure 3. CD spectra of L-alanyl-L-tyrosine doped SiO2/PPy before (a) and after (b) hydrolysis, and L-Ala and L-Tyr co-imprinted SiO2/PPy (c). XRD Pattern of L-Ala and L-Tyr Co-Imprinted SiO2/PPy. The XRD patterns of SiO2, PPy, dipeptide doped SiO2/PPy as well as L-Ala and L-Tyr co-imprinted SiO2/PPy are shown in Figure 4. The broad peak centered at around 2θ = 23.5° is characteristic of amorphous SiO2 (curve a),34 and the broad peak located at around 2θ = 25.1° confirms the amorphous structure of PPy (curve b).35 For the dipeptide doped SiO2/PPy, only one broad peak is observed at around 2θ = 24.0° (curve c), which might be due to the superposition of the characteristic peaks of SiO2 and PPy, and this diffraction peak is still observed on the XRD pattern of the co-imprinted SiO2/PPy after being hydrolyzed and washed with alcohol and acetic acid (curve d). This result indicates that the hydrolysis of the dipeptide and the removal of L-Ala and L-Tyr almost do not influence the crystal structure of the material.


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a

Intensity / a.u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b c d

0

20

40

60

80

2 theta / Degree

Figure 4. XRD patterns of SiO2 (a), PPy (b), L-alanyl-L-tyrosine doped SiO2/PPy (c), and L-Ala and L-Tyr co-imprinted SiO2/PPy (d). Morphologies of L-Ala and L-Tyr Co-Imprinted SiO2/PPy. The morphologies of SiO2, dipeptide doped SiO2/PPy and co-imprinted SiO2/PPy are shown in Figure 5. As can be seen, the as-prepared SiO2 consists of nanospheres with a uniform size of ~300 nm (Figure 5A), which is then highly coated by PPy after in situ pyrrole polymerization/dipeptide doping process (Figure 5B and D). After being hydrolyzed in HCl solution and washed with the mixture of alcohol and acetic acid to remove the produced L-Ala and L-Tyr, the PPy coating on the co-imprinted SiO2/PPy is significantly reduced (Figure 5C and E), which might be ascribed to the fact that a certain amount of PPy coating is lossed during the hydrolysis and subsequent washing process.



(A)

(B)

(C)

(D)

(E)

Figure 5. FESEM images of SiO2 (A), dipeptide doped SiO2/PPy (B) and co-imprinted SiO2/PPy (C). TEM images of dipeptide doped SiO2/PPy (D) and co-imprinted SiO2/PPy (E). EIS of L-Ala and L-Tyr Co-Imprinted SiO2/PPy. Figure 6 shows the EIS of GCE modified with co-imprinted SiO2/PPy, dipeptide doped SiO2/PPy as well as unmodified GCE in 0.1 M KCl solution containing 5 mM [Fe(CN)6]3–/4–, and the EIS was carried out at the open circuit potential of 0.24 V with a sine wave potential of 5 mV in the frequency range from 105 to 0.01 Hz. The obtained Nyquist plots consist of a suppressed semicircle in the high frequency region and a straight line in the low frequency region. Usually, the diameter of the suppressed semicircle refers to the charge transfer resistance (Rct) at the 12 ACS Paragon Plus Environment

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electrode-solution interface.36 As can be seen, GCE modified with dipeptide doped SiO2/PPy exhibits a remarkably higher Rct (curve b, 153.8 ) compared with unmodified GCE (curve a, 55.7 ), which can be ascribed to the low conductivity of dipeptide doped SiO2/PPy. Interestingly, the Rct of the GCE modified with co-imprinted SiO2/PPy is decreased again to 121.0  (curve c). The doped L-alanyl-L-tyrosine is a dipeptide with poor conductivity, which is against charge transfer, and therefore the Rct of the GCE modified with co-imprinted SiO2/PPy is decreased a little after the doped dipeptide is hydrolyzed and the produced L-Ala and L-Tyr are removed. The EIS result further confirms successful hydrolysis of the doped dipeptide and the removal of the hydrolysates (L-Ala and L-Tyr). 800

Rs

700

Z-imaginary / ohm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Q

600

R ct W d

500

a

400 300 c

200

b

100 0 0

100

200

300

400

500

600

700

800

Z-real / ohm

Figure 6. Nyquist plots of unmodified GCE (a), GCE modified with dipeptide doped SiO2/Ppy (b) and co-imprinted SiO2/PPy (c) in 0.1 M KCl containing 5 mM [Fe(CN)6]4–/3–. Inset is the equivalent circuit, where Rs, Rct, Wd and Q represent solution resistance, charge transfer resistance, Warburg resistance and constant phase elements, respectively. Simultaneous Chiral Recognition of D/L-Ala and D/L-Tyr. Next, the practicality of the co-imprinted chiral sensor is evaluated by simultaneous chiral recognition of D/L-Ala and D/L-Tyr. The co-imprinted chiral sensor is immersed into 25 mL 0.1 M PBS (pH = 7.0) containing L-amino acids (2 mM L-Ala and 1 mM L-Tyr) or D-amino acids (2 mM D-Ala 13 ACS Paragon Plus Environment


and 1 mM D-Tyr), and then a positive potential of +0.3 V is applied at the chiral sensor for 1000 s for the rebinding of L- and D-amino acids. The DPVs of the L- and D-amino acids rebound to the co-imprinted sensor are shown in Figure 7A. Obviously, the peak currents of L-amino acids are greatly higher than those of D-amino acids (IL-Ala/ID-Ala = 1.93, IL-Tyr/ID-Tyr = 1.75), suggesting that the L-Ala and L-Tyr co-imprinted chiral sensor exhibits higher affinity toward L-amino acids than D-amino acids. Such specificity for L-amino acids can be ascribed to the “morphology-memory” effect of molecular imprinting. For a comparison, simultaneous recognition of D/L-Ala and D/L-Tyr is also investigated at the NI-SiO2/PPy modified GCE under the same conditions, and the results of DPVs indicate that both Ala and Tyr enantiomers can hardly be effectively recognized at the NI-SiO2/PPy modified GCE (Figure 7B). In fact, the use of multiple L-amino acids including L-Ala as the direct templates has also been studied and reported in our group,25 and the obtained recognition efficiency of D/L-Ala is a little lower than that reported in this work (IL-Ala/ID-Ala = 1.89 versus IL-Ala/ID-Ala = 1.93). This comparison clearly suggests that our co-imprinted chiral sensor can be used for efficient recognition of amino acids enantiomers. 3.0

5.0 4.5

(A)

L-Tyr

4.0

(B)

L-Tyr D-Tyr

2.5

3.5

Current / µA

Current / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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3.0 2.5

L-Ala

2.0 1.5

2.0 L-Ala

1.5

D-Ala

D-Ala

1.0

D-Tyr

0.5 -0.8

-0.4

0.0

0.4

0.8

1.0 -0.8

1.2

-0.4

0.0

0.4

0.8

1.2

Potential / V (vs. Ag/AgCl)

Potential / V (vs. Ag/AgCl)

Figure 7. Differential pulse voltammograms of 2 mM L-Ala, 1 mM L-Tyr, 2 mM D-Ala and 1 mM D-Tyr rebound to GCE modified with L-Ala and L-Tyr co-imprinted SiO2/PPy (A) and 14 ACS Paragon Plus Environment

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NI-SiO2/PPy (B) in 0.1 M PBS of pH 7.0. Optimization of Rebinding Potential and Rebinding Time. Since the isoelectric points (PI) of Ala and Tyr are 6.0237 and 5.66,38 respectively, Ala and Tyr are negatively charged in 0.1 M PBS of pH 7.0. Therefore, the rebinding of L-Ala and L-Tyr can be facilitated when a positive potential is applied at the co-imprinted chiral sensor. Figure 8 shows the influence of rebinding potential on the recognition efficiency of D/L-Ala and D/L-Tyr at the co-imprinted chiral sensor. It shows that the recognition efficiency of D/L-Tyr is gradually increased with increasing potential from 0.1 to 0.5 V, which is due to the increasing electrostatic attractions between the sensor and Tyr. However, the recognition efficiency of D/L-Ala is remarkably decreased when the potential is higher than 0.3 V. As can be seen from Figure 7, the oxidation peak potentials of Tyr and Ala appear at ~0.8 V and ~0.2 V, respectively. The Tyr enantiomers can exist stably in the potential region from 0.1 to 0.5 V, whereas the severe oxidation of Ala beyond 0.3 V will greatly deteriorate the recognition efficiency of D/L-Ala. In order to achieve high recognition efficiency for both D/L-Ala and D/L-Tyr, the rebinding potential is set at 0.3 V throughout the experiments.

Peak current ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.95

IL-Ala / ID-Ala

1.90

IL-Tyr / ID-Tyr

1.85 1.80 1.75 1.70 1.65 1.60 0.1

0.2

0.3

0.4

0.5

Rebinding potential / V (vs. Ag/AgCl)

Figure 8. Influence of rebinding potential on the recognition efficiency of D/L-Ala and 15 ACS Paragon Plus Environment


D/L-Tyr at the co-imprinted chiral sensor. Error bars represent standard deviations for three independent measurements. The rebinding time is also optimized, and the results are shown in Figure 9. It shows that the recognition efficiency for both D/L-Ala and D/L-Tyr is significantly increased with increasing time till 1000 s, while it is slightly decreased with further increased time. This result might be ascribed to the fact that the rebinding of L-Ala and L-Tyr to the co-imprinted SiO2/PPy reaches the saturation at the time of 1000 s, and then the non-specific adsorption of Ala and Tyr on the surface of the co-imprinted SiO2/PPy would lead to the slightly decreased recognition efficiency.39

Peak current ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2.1

IL-Ala / ID-Ala

2.0

IL-Tyr / ID-Tyr

1.9 1.8 1.7 1.6 1.5 1.4 1.3 200

400

600

800

1000

1200

1400

Rebinding time / s

Figure 9. Influence of rebinding time on the recognition efficiency of D/L-Ala and D/L-Tyr at the co-imprinted chiral sensor. Error bars represent standard deviations for three independent measurements. CONCLUSIONS In summary, a novel multi-imprinted chiral sensor is fabricated via the hydrolysis of L-alanyl-L-tyrosine dipeptide doped in SiO2/PPy in HCl aqueous solution and subsequent removal of the produced L-Ala and L-Tyr. The resultant L-Ala and L-Tyr co-imprinted 16 ACS Paragon Plus Environment

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SiO2/PPy is then applied in the simultaneous chiral recognition of D/L-Ala and D/L-Tyr. Compared with NI-SiO2/PPy, the enantiomers of Ala and Tyr can be well recognized at the co-imprinted chiral sensor. Both the rebinding potential and the rebinding time are optimized in this work. Such co-imprinting strategy might have a great potential in the development of single-template molecularly imprinted chiral sensor for simultaneous recognition of multiple chiral compounds, which is especially useful in the analysis of complicated chiral systems. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel.: 86-519-86330253. Fax: 86-519-86330167. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This research was supported financially by National Natural Science Foundation of China (21775013, 21804013), Natural Science Foundation of Jiangsu Province (BK20171194), Advanced

Catalysis

and

Green

Manufacturing

Collaborative

Innovation

(ACGM2016-06-27) and Qing Lan Project of Jiangsu Higher Education Institutions.


Center


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