Covalent Functionalization of Bovine Serum Albumin with Graphene

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Covalent Functionalization of Bovine Serum Albumin with Graphene Quantum Dots for Stereospecific Molecular Recognition Qiumin Ye, Lili Guo, Datong Wu, Baozhu Yang, Yongxin Tao, Linhong Deng, and Yong Kong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02605 • Publication Date (Web): 15 Aug 2019 Downloaded from pubs.acs.org on August 15, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

Covalent Functionalization of Bovine Serum Albumin with Graphene

Quantum

Dots

for

Stereospecific

Molecular

Recognition

Qiumin Ye, Lili Guo, Datong Wu, Baozhu Yang, Yongxin Tao, Linhong Deng, 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: Stereospecific molecular recognition with simple and easily available proteins is of significant importance in life science and biomaterial science. Herein, we report on a chiral sensing platform, graphene quantum dots (GQDs) functionalized bovine serum albumin (BSA), for chiral recognition of tryptophan (Trp) isomers. Amidation reaction between BSA and GQDs was directly responsible for the introduction of GQDs to BSA, resulting in significant changes in the spatial configuration of BSA and the exposure of more chiral sites at the protein surface. The BSA-GQDs based chiral sensor exhibited good biomolecular homochirality in the recognition of Trp isomers, and the higher affinity of BSA-GQDs toward L-Trp than its isomer, D-Trp, was also revealed by density functional theory (DFT) considering the possible hydrogen bonds between the Trp isomers and the solvent accessible residues of BSA.


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Chirality (handedness) is an interesting phenomenon in nature. As the molecular building blocks of life, amino acids are all chiral molecules except for glycine, and the isomers of amino acids exhibit different biological activities.1‒3 Although various strategies such as molecular imprinting,4,5 ligand exchanging6,7 and supramolecular interactions8 have been proposed for stereospecific molecular recognition, chiral recognition of amino acids is still considered to be one of the most difficult issues due to the similar physicochemical properties of the optical isomers.9,10 Recently, the combination of electrochemistry and chiral recognition has opened up a new avenue for stereospecific molecular recognition.11 In particular, electrochemistry can convert a chiral recognition event into discernible changes in electrochemical signals,12‒15 making it an ideal technology for isomers recognition. The key of electrochemical chiral recognition is the match between the isomers to be identified and the electrode modifier, similar to hands and gloves. Most biological macromolecules such as proteins, nucleic acids and natural polysaccharides possess intrinsic chirality and exhibit fascinating stereospecific behavior. Particularly, proteins composed of chiral subunits (L-amino acids) are capable to bind small molecules and these bind interactions can be stereospecific, and thus proteins have been immobilized as chromatographic support for chiral resolution.16,17 However, as far as we are aware, little or no attention has been paid to develop protein-based electrochemical chiral sensors, which might be due to the shielding of chiral sites in proteins and the consequently low recognition efficiency of proteins. Herein, we first report on the covalent functionalization of bovine serum albumin (BSA) with graphene quantum dots (GQDs) for chiral recognition of the isomers of tryptophan 3 ACS Paragon Plus Environment


(Trp), an essential amino acid in most biological systems. BSA is selected as the model protein not only because of its low cost, easy availability and medical importance, but also because of its three helical domains (I, II, and III), which are arranged in the shape of a heart and directly responsible for the stereoselectivity.18‒20 The applications of BSA and its nanocomposites for electrochemical and fluorescent biosensors have been reported very recently.21‒23 GQDs of small sizes have carboxylic acid moieties at the edge which contribute to their excellent biocompability.24 More importantly, the oxygen-containing groups on the surface of GQDs impart them with suitability for functionalization with various biological species.25 In this work, the surface functionalization of BSA with GQDs is achieved via the amidation reaction between BSA and GQDs. Of particular interest is that the original morphology of BSA is remarkably changed after the introduction of GQDs, leading to sufficient exposure of the chiral sites at the surface of BSA. As a result, excellent biomolecular homochirality in the recognition of the Trp isomers is achieved with the BSA-GQDs based chiral sensor.


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EXPERIMENTAL SECTION Reagents and Apparatus. Bovine serum albumin (BSA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride, D-tryptophan (D-Trp, 99%) and L-tryptophan (L-Trp, 99%) were purchased from Aladdin Chemistry Co., Ltd (Shanghai, China). Citric acid monohydrate, potassium ferricyanide (K3Fe(CN)6), potassium ferrocyanide (K4Fe(CN)6) and other chemicals not mentioned were received from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All aqueous solutions were prepared with ultrapure water (Milli-Q, Millipore). The morphologies of BSA and BSA-GQDs were characterized with a Supra55 field emission scanning electron microscope (FESEM, Zeiss, Germany). The Fourier transform infrared (FT-IR) spectra of GQDs, BSA and BSA-GQDs were recorded with a Nicolet FTIR-8400S spectrophotometer (Shimadzu, Japan) using KBr pellets. The UV spectra of different samples were recorded on a model U-3900 UV-vis spectrophotometer (Hitachi, Japan). All electrochemical measurements were carried out in a conventional three-electrode cell connected to a CHI-660E electrochemical workstation. The counter electrode was a platinum plate (10  5 mm), and a KCl saturated calomel electrode (SCE) was taken as the reference electrode. A glassy carbon electrode (GCE, 3 mm in diameter) or a modified GCE (BSA-GQDs/GCE, GQDs/GCE or BSA/GCE) was used as the working electrode. Hydrophilicity of different samples was evaluated with a water contact angle goniometer (model DSA25, Kruss GmbH, Germany), and a water droplet (3 μL) was dropped onto the sample surface for each testing. To reveal the mechanisms of chiral recognition, the molecular dynamics (MD) simulation was accomplished with a Gaussian 09 software 5 ACS Paragon Plus Environment


package. Preparation of GQDs. GQDs were prepared via direct pyrolysis of citric acid.26,27 In a typical procedure, 3.5 g of citric acid monohydrate was put into a ceramic crucible and heated to 200 °C in an electric furnace. After 30 min, the ceramic crucible was cooled to room temperature (25 °C), and the obtained yellow solids (GQDs) were dissolved in 10 mL of 0.01 M phosphate buffer saline (PBS, pH = 7.4) by ultrasonification. Preparation of BSA-GQDs via Amidation Reaction. 4 mg of BSA was dissolved in 1 mL of 0.01 M PBS (pH = 7.4) by ultrasonification, resulting the aqueous solution of BSA. Since the isoelectric point (PI) of BSA is 4.7,28 the BSA dissolved in PBS of pH 7.4 is negatively charged. 5 mL of the GQDs solution was added into a 25 mL beaker, and then the pH of the GQDs solution was adjusted to 5.0 to protonate the carboxyl groups of GQDs29 and avoid the electrostatic repulsion between GQDs and BSA. Next, 80 mg EDC and 80 mg NHS were added to the GQDs solution with magnetic stirring for 30 min to activate the carboxyl groups of GQDs. After that, 50 μL of the BSA solution (4 mg mL–1) was added into the solution of activated GQDs with continuous stirring for 24 h, and the composites of BSA-GQDs were produced via the amidation reaction between the –NH2 groups of BSA and the –COOH groups of GQDs. As BSA has a large molecular weight of 66 kDa,18 the liquid containing BSA-GQDs was transferred into a dialysis bag with molecular weight cut off 70 kDa and dialyzed in water for 3 days to remove un-reacted BSA, GQDs and activators (EDC and NHS). Finally, the dialysis bag was taken out and blow-dried with an electric fan until 1 mL of the pale yellow BSA-GQDs solution was left, which was stored at 4 °C prior to use. Recognition of Trp Isomers with BSA-GQDs Chiral Sensor. The BSA-GQDs/GCE 6 ACS Paragon Plus Environment

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was prepared by a simple casting method. Before casting, the GCE was polished with 0.05 μm alumina slurries and rinsed thoroughly with HNO3-H2O (1:1 by volume), ethanol and water. After that, 5 μL of the BSA-GQDs solution was cast onto the surface of the pre-treated GCE, which was then incubated at 4 °C for 24 h. For control experiments, GQDs/GCE and BSA/GCE were also prepared by the same procedures using GQDs and BSA instead of BSA-GQDs, respectively. Stereospecific molecular recognition of Trp isomers was investigated by differential pulse voltammetry. The three-electrode cell with the BSA-GQDs/GCE as the working electrode was immersed into 25 mL of 0.1 M PBS (pH = 7.0) containing 0.5 mM L-Trp or D-Trp for 60 s, and then the differential pulse voltammograms (DPVs) of L-Trp and D-Trp combined with the BSA-GQDs were recorded. Both the peak current ratio of L-Trp to D-Trp (IL-Trp/ID-Trp) and the peak potential separation (ΔEp = ED-Trp − EL-Trp) were used as the indicator to evaluate the recognition efficiency of the BSA-GQDs/GCE. The as-fabricated chiral sensor can be repeatedly used, however, activation of the sensor is needed. To avoid the interferences from the residual Trp in the next measurement, the sensor was activated by cyclic voltammetry since the residual Trp can be oxidized irreversibly by cyclic voltammetry. Specifically, the chiral sensor was activated by cyclic voltammetry in 0.1 M PBS of pH 7.0 for 20 cycles (0.4 ~ 1.2 V, 100 mV s–1) to restore its activity. RESULTS AND DISCUSSION Morphologies of BSA and BSA-GQDs. We examine the morphologies of BSA and BSA-GQDs (Figure 1). BSA exhibits a regular but dense surface, which is definitely disadvantageous for the accommodation of guest molecules. After the functionalization with 7 ACS Paragon Plus Environment


GQDs, amounts of cavities are observed within the resultant BSA-GQDs, and the formation of such cavities is most likely due to the fact that the peptide strands of BSA are greatly extended via the amidation reaction between the –COOH groups of GQDs and the –NH2 groups of BSA (Figure 2). More importantly, more chiral sites within the BSA are exposed at the protein surface after the functionalization with GQDs owing to the significant changes in the spatial configuration of BSA, leading to decreased hydrophobility of the BSA-GQDs composites and easy accessibility to the guest molecules.

(B)

(A)

Figure 1. SEM images of BSA (A) and BSA-GQDs composites (B).

Figure 2. Illustration showing the amidation reaction between BSA and GQDs. Characterizations of BSA-GQDs. The BSA-GQDs interaction is further studied by FT-IR and UV spectra. As shown in the FI-IR spectra (Figure 3), the protein amide I band at 1658 cm–1 and amide II band at 1539 cm–1 are observed for BSA,30,31 and the bands at 1766 and 1718 cm–1 can be assigned to the antisymmetric and symmetric stretching of C=O of GQDs.32 Slight shift of these characteristic bands occurs for BSA-GQDs. For example, the 8 ACS Paragon Plus Environment

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bands for GQDs at 1766 and 1718 cm–1 are downshifted to 1764 and 1714 cm–1 for the BSA-GQDs composites, meanwhile, the band for BSA at 1539 cm–1 is shifted to 1542 cm–1, indicating the formation of amide bond (–NH---CO–) between GQDs and BSA. Noted that the band for BSA at 1658 cm–1 is also red-shifted to 1652 cm–1, and this shift is indicative of the conformational change of the α-helix of BSA31 after the functionalization with GQDs, agreeing well with the SEM result. The structural changes of BSA by GQDs functionalization are also confirmed by the UV spectra (Figure 4). For GQDs, there is no clear absorption peak but a long absorption edge, which is in good agreement with the previous report.33 A strong absorption band of BSA at around 278 nm is observed, which can be ascribed to the three amino acid residues (tryptophan, tyrosine and phenylalanine) in BSA.34 The band is moderately shifted toward longer wavelength (332 nm) on the spectrum of BSA-GQDs, indicating that with the incorporation of GQDs, the peptide strands of BSA molecules are extended. This result agrees well with the previous report,35 in which the absorption band of BSA also shows moderate shifts after the addition of fluoroquinolones. BSA

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


1539 1658

GQDs

1766 1718

BSA-GQDs

1542 1764 1652 1714

3500

3000

2500

2000

1500

-1

1000

Wavenumber / cm

Figure 3. FT-IR spectra of BSA, GQDs and BSA-GQDs composites.


500


BSA GQDs BSA-GQDs

1.5

Absorbance

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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1.2 0.9 0.6 0.3

278 332

0.0 250

300

350

400

Wavelength / nm

Figure 4. UV spectra of BSA, GQDs and BSA-GQDs composites. Electrochemical Properties of BSA-GQDs. The electrochemical properties of the BSA-GQDs/GCE as well as other modified electrodes are studied. Figure 5A shows the cyclic voltammograms (CV) of different samples in 0.1 M KCl containing [Fe(CN)6]4–/3– couple as the redox probe. Due to the redox reaction between [Fe(CN)6]4– and [Fe(CN)6]3–, there is a pair of well-defined redox peaks at bare GCE. The peak currents are decreased a little at the GQDs/GCE, and this phenomenon might be ascribed to the abundant electronegative groups (–COOH, –OH) on GQDs that hamper the electron transfer of [Fe(CN)6]4–/3– to the electrode surface.36 The peak currents are remarkably decreased at the BSA/GCE, which could be attributed to the following two aspects: (1) As a kind of protein, BSA has poor conductivity, which is against the electron transfer; (2) the pI of BSA is 4.7,28 and BSA is negatively charged in 0.1 M KCl (pH ~7.0) and the electrostatic repulsions between BSA and [Fe(CN)6]4–/3– lead to greatly decreased current at the BSA/GCE. Noted that the peak currents at the BSA-GQDs/GCE are a little higher than those at BSA/GCE, which might be due to the fact that amounts of cavities exist within the BSA-GQDs (Figure 1B), facilitating the electron transfer to the electrode interface. The results of electrochemical 10 ACS Paragon Plus Environment

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impedance spectroscopy (EIS) are in good agreement with those of CV. As shown in Figure 5B, the charge transfer resistance (Rct), which is indicated by the diameter of the suppressed semicircle, of BSA-GQDs/GCE is still a little smaller than that of BSA/GCE (1927 Ω versus 2606 Ω). 5000

(A)

a

100

b d c

50

Z-imaginary / ohm

150

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


0 -50

-100 -150

(B)

Rs

Q

4000

Rct

3000 2000

a

Wd

d

b

c

1000 0

-0.2

0.0

0.2

0.4

0.6

0

Potential / V (vs. SCE)

1000

2000

3000

Z-real / ohm

4000

5000

Figure 5. Cyclic voltammograms (A) and Nyquist plots (B) of GCE (a), GQDs/GCE (b), BSA/GCE (c) and BSA-GQDs/GCE (d) in 0.1 M KCl containing 5 mM [Fe(CN)6]4–/3–. Scan rate of CV: 100 mV s–1; frequency range of Nyquist plots: 106 to 0.01 Hz. Inset of (B): the equivalent circuit. Chiral Recognition of Trp Isomers with BSA-GQDs. Next, we evaluate the capability of different samples for the recognition of Trp isomers by differential pulse voltammetry, and the results are shown in Figure 6. As previously reported, the electrode reaction of Trp is totally irreversible, and two electrons and two protons transfer is involved in the electrode reaction process of Trp.37 Obviously, bare GCE is incapable of recognizing the Trp isomers at all since the DPVs of L- and D-Trp are completely overlapped (Figure 6A), which can be ascribed to the absence of chiral sites on the bare GCE. Because the oxygen-containing groups of GQDs can form hydrogen bonds with the carboxyl or hydroxyl groups of Trp, the oxidation peak currents of the Trp isomers are greatly increased at the GQDs/GCE (Figure 11 ACS Paragon Plus Environment


6B). However, the recognition ability of the GQDs/GCE is still rather poor owing to the few chiral sites on GQDs. When the BSA/GCE is used for the recognition of Trp isomers, discernable differences in both peak currents and peak potentials can be clearly observed (Figure 6C), implying that the intrinsic chirality of BSA plays a crucial role in chiral recognition. The recognition efficiency indicated by peak current ratio (IL/ID) and peak potential separation (ΔEp) is remarkably increased at the BSA-GQDs/GCE (Figure 6D), and the enhanced recognition ability of the BSA-GQDs might be ascribed to the fact that the peptide strands of BSA are greatly extended by the incorporation of GQDs and consequently more chiral sites shielded in BSA are exposed on the protein surface due to the significant changes in the spatial configuration of BSA. The value of IL/ID at the BSA-GQDs/GCE is 3.67, which outperforms those obtained at -cyclodextrin (-CD)/GCE (2.30),11 sulfonated chitosan (CS)/GCE (2.38)38 and Cu2+-modified -CD/GCE (3.28)10 when used for the recognition of Trp isomers. The possible reason might be attributed to the fact that the chiral sites of -CD are surrounded by its hydrophobic cavity and the chiral sites of CS are surrounded by its polymer chains, while the chiral sites at the surface of BSA are sufficiently exposed to the Trp isomers after the introduction of GQDs.


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6

(A)

L-Trp

Current / μA

Current / μA

IL/ID = 1.0 △EP = 0 mV

2

(B)

L-Trp

5

D-Trp

3

D-Trp

4

IL/ID = 1.02 △EP = 4 mV

3 2

1

1 0

0.4

0.5

0.6

0.7

0.8

0.9

0

1.0

0.4

0.8

1.6

(C)

L-Trp D-Trp

0.4

IL/ID = 1.93 △EP = 44 mV

0.2

0.0

0.4

0.6

0.8

1.0

0.6

0.7

0.8

0.9

1.2

(D)

1.0

L-Trp D-Trp

1.2

Current / μA

0.6

0.5



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


IL/ID = 3.67 0.8

△EP = 96 mV

0.4

0.0

0.4

0.6

0.8

1.0

1.2



Figure 6. Differential pulse voltammograms of 0.5 mM L-Trp and D-Trp combined with bare GCE (A), GQDs/GCE (B), BSA/GCE (C) and BSA-GQDs/GCE (D) in 0.1 M PBS of pH 7.0. Temperature: 25 oC. Higher Affinity of BSA-GQDs toward L-Trp. Perhaps more important is that we further discover a higher peak current of L-Trp at both BSA-GQDs/GCE and BSA/GCE, suggesting that BSA exhibits a higher affinity toward L-Trp than D-Trp. That is to say, BSA is preferably incubated with L-Trp compared with its isomer, which can be revealed by density functional theory (DFT) considering the possible hydrogen bonds between the Trp isomers and the solvent accessible residues of BSA. Figure 7 shows the sequence alignment of BSA, in which the sixteen residues with a solvent accessible area ≤ 10 Å20 are marked in red. For simplicity, the sixteen solvent accessible residues are divided into fifteen segments (marked with blue line), which are Asp-Thr-His (DTH), Glu-Ile-Ala (EIA), Lys-Gly-Leu (KGL), Phe-Ser-Gln (FSQ), Trp-Gly-Lys (WGK), Thr-Met-Arg (TMR), Lys-Val-Leu (KVL), 13 ACS Paragon Plus Environment


Ala-Leu-Lys (ALK), Trp-Ser-Val (WSV), Phe-Ala-Glu (FAE), Tyr-Ser-Arg (YSR), Tyr-Ala-Val-Ser-Val-Leu (YAVSVL), Lys-Leu-Lys (KLK), Leu-Ile-Val (LIV) and Leu-Leu-Lys (LLK), respectively. To understand the spatial constraints during the combination of Trp isomers with the fifteen segments, the structure of BSA is deposited in the Protein Data Bank (PDB, code: 3V03) by using the PyMOL Molecular Graphics System.39 It shows that the incubation of L-/D-Trp in these segments is hardly affected by the spatial configuration of the secondary structure of these BSA residues (Figure S1), i.e., hydrogen bonds might be the crucial factor affecting the incubation of Trp isomers in BSA.

Figure 7. Sequence alignment of BSA. Specifically, the sixteen residues with a solvent accessible area ≤10 Å are marked in red, which are divided into fifteen segments marked with blue line. Abbreviations: Aspartic acid-D, threonine-T, histidine-H, lysine-K, serine-S, glutamic acid-E, isoleucine-I, alanine-A, arginine-R, phenylalanine-F, leucine-L, glycine-G, valine-V, glutamine-Q, tyrosine-Y, cysteine-C, praline-P, asparagines-N, methionine-M, 14 ACS Paragon Plus Environment

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tryptophan-W. To obtain further information regarding bond length and bond angle of these hydrogen bonds between the solvent accessible segments and the Trp isomers, the ground state structures of the complexes of the fifteen segments and L-/D-Trp are optimized by DFT without symmetry constrains,40,41 and the MD simulation is carried out with the Gaussian 09 software package. By using the 6-311G (d,p) basis sets for N, O, C, H and S atoms, the obtained results are presented in Figure 8. According to the range of bond length and bond angle, hydrogen bond can be classified into three categories including very strong, strong and weak hydrogen bond (Table 1),42 and a comprehensive comparison of the data obtained from Figure 8 is listed in Table 2. Although some of the solvent accessible segments (DTH, FSQ, ALK, WSV, YSR, KLK) exhibit higher affinity toward D-Trp than L-Trp, most of the segments (EIA, KGL, WGK, TMR, KVL, FAE, YAVSVL, LIV, LLK) can form stable hydrogen bonds with L-Trp compared with its isomer. Therefore, BSA is preferably incubated with L-Trp owing to the favourable hydrogen bonds between them.




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Figure 8. Optimized ground state structures of the complexes of the fifteen solvent accessible segments and the Trp isomers.

Table 1. Bond length and bond angle range of very strong, strong and weak hydrogen bond. Very strong

Project

H-bond

Strong H-bond

Weak H-bond

Bond length range / Å

1.2 ~ 1.5

1.5 ~ 2.2

2.2 ~ 3.0

Bond angle range / °

175 ~ 180

130 ~ 175

90 ~ 180

Table 2. Comprehensive comparison of bond length and bond angle of all the hydrogen bonds between the solvent accessible segments and the Trp isomers. Bond length and bond angle of all the hydrogen bonds 1 DTH/L-Trp

2 None

1

2

DTH/D-Trp

2.04 Å, 168.56°

2.26 Å, 128.66°

EIA/L-Trp

2.07 Å, 155.24°

2.83 Å, 167.79°

EIA/D-Trp

2.27 Å, 144.82°

KGL/L-Trp

2.18 Å, 144.96°

1.98 Å, 172.54°

KGL/D-Trp

2.08 Å, 136.03°

3: 2.19 Å ,155.21° FSQ/L-Trp

2.30 Å, 135.90°

WGK/L-Trp

2.52 Å, 144.20°

2.12 Å, 140.81°

3: 2.29 Å, 161.58°

2.44 Å, 136.57°

FSQ/D-Trp

2.54 Å, 141.81°

WGK/D-Trp

2.38 Å, 149.98°


2.24 Å, 119.84°


TMR/L-Trp

1.88 Å, 154.76°

KVL/L-Trp

2.72 Å, 124.96°

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TMR/D-Trp

2.50 Å, 124.57°

2.18 Å, 126.89°

KVL/D-Trp

2.58 Å, 102.01°

ALK/L-Trp

2.26 Å, 154.25°

ALK/D-Trp

2.21 Å, 141.17°

2.00 Å, 179.67°

WSV/L-Trp

2.16 Å, 151.08°

2.24 Å, 132.53°

WSV/D-Trp

2.23 Å, 136.18°

2.06 Å, 169.97°

FAE/L-Trp

2.49 Å, 132.84°

2.25 Å, 125.41°

FAE/D-Trp

2.65 Å, 101.73°

2.23 Å, 151.84°

YSR/L-Trp

2.23 Å, 148.04°

YSR/D-Trp

2.25 Å, 150.88°

2.36 Å, 119.18°

YAVSVL/L-Tr

2.30 Å, 147.23°

YAVSVL/D-Tr

2.82 Å, 108.31°

2.17 Å, 136.50°

2.18 Å, 131.42°

p

p 3: 2.22 Å, 135.81° 4: 2.19 Å, 131.18°

3: 2.83 Å, 132.57° 4: 2.41 Å, 118.45°

5: 2.38 Å, 156.52° 6: 2.21 Å, 132.11°

5: 2.33 Å, 124.28°

KLK/L-Trp

2.16 Å, 162.66°

2.76 Å, 172.03°

KLK/D-Trp

LIV/L-Trp

2.09 Å, 158.52°

2.50 Å, 127.60°

LIV/D-Trp

LLK/L-Trp

2.13 Å, 130.78°

2.07 Å, 162.35°

LLK/D-Trp

2.02 Å, 148.29°

2.09 Å, 147.81°

None 2.01 Å, 156.49°

2.34 Å, 125.32°

The nine solvent accessible segments showing higher affinity toward L-Trp are marked in red.

The higher affinity of BSA toward L-Trp can be further confirmed by comparing the UV spectra of the Trp isomers before and after their combination with BSA-GQDs. Actually, Land D-Trp are identical in absorption intensity (Figure 9), which can be ascribed to the same functional groups of the two isomers. After the incubation of L- and D-Trp in the BSA-GQDs for 60 s, the maximum absorption (279 nm) of the residual L-Trp is decreased about 18% (Figure 9A), which is more pronounced than that of the residual D-Trp (about 10%, Figure 9B). The UV results also clearly indicate that BSA exhibits higher affinity toward L-Trp than D-Trp.


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3.0

L-Trp

(A)

L-Trp +

3.0

BSA-GQDs

2.5 2.0 1.5 1.0 0.5 0.0 240

D-Trp

(B)

D-Trp + BSA-GQDs

2.5

Absorbance

Absorbance

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


2.0 1.5 1.0 0.5

260

280

300

320

340

Wavelength / nm

0.0 240

260

280

300

320

340

Wavelength / nm

Figure 9. UV spectra of 0.5 mM L-Trp (A) and D-Trp (B) before and after their combination with BSA-GQDs for 60 s. The successful functionalization of BSA with GQDs and the higher affinity of BSA toward L-Trp are indicated by the hydrophilicity evaluation (Figure 10, Table S1). The water contact angle on BSA is 42.4°, however, it is greatly decreased to 34.8° after the functionalization with GQDs. The enhanced wettability of BSA-GQDs can be attributed to the incorporation of GQDs with polar functional groups (−COOH). It is noteworthy that the water contact angle on BSA-GQDs is further decreased to 27.3° after the incubation of L-Trp, which is more pronounced than that after the incubation of D-Trp (30.2°). The Trp isomers are highly hydrophilic due to their polar functional groups such as −COOH and −NH2, and therefore the smaller water contact angle on BSA-GQDs/L-Trp suggests that more L-Trp is incubated in the BSA-GQDs than D-Trp. In other words, the BSA-GQDs has good biomolecular homochirality and exhibits higher affinity toward L-Trp than its isomer.



42.4 ± 0.7°

34.8 ± 1.0°

BSA-GQDs

BSA

30.2 ± 0.8°

27.3 ± 0.8°

BSA-GQDs/D-Trp

BSA-GQDs/L-Trp

Figure 10. Water droplet (3 μL) images and water contact angle measurements on different samples. Finally, quantitative estimation of the Trp isomers in non-racemic mixtures is studied. As can be seen, there is a broad oxidation peak at the BSA-GQDs/GCE, and the peak current is decreased with increasing D-Trp% in the mixture (Figure 11A). The calibration plot of peak current versus D-Trp% exhibits a good linearity (Figure 11B), suggesting that the as-fabricated BSA-GQDs/GCE has the potential to be a reliable chiral sensor.

1.4

1.6

(A) 1.2

D-Trp 100%

0.8

0.4

(B)

1.2

D-Trp 0%

Peak 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

Page 20 of 29

1.0 0.8 0.6 y = -0.0063 x + 0.9523 R2 = 0.9016

0.4 0.2

0.0

0.4

0.6

0.8

1.0


0

1.2


20

40 60 D-Trp%

80

100

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Figure 11. (A) Differential pulse voltammograms of Trp isomers (total concentration, 0.5 mM) with different D-Trp% (0%, 20%, 40%, 60%, 80%, and 100%) at the BSA-GQDs/GCE in 0.1 M PBS of pH 7.0 at 25 oC. (B) Calibration plot of peak current versus D-Trp%. Error bars represent the standard deviation for three independent measurements. CONCLUSIONS To summarize, we demonstrate that the functionalization of BSA with GQDs via amidation interaction can construct a chiral sensor that is preferable to L-Trp. The incorporation of GQDs can result in significant changes in the spatial configuration of BSA and the sufficient exposure of more chiral sites at the protein surface, as revealed by the SEM results. The BSA-GQDs composites are then used to modify the GCE and the resultant chiral sensor exhibits good biomolecular homochirality in the recognition of Trp isomers. Also in this work, the higher affinity of BSA-GQDs toward L-Trp than its isomer, D-Trp, is also explained by DFT considering the possible hydrogen bonds between the Trp isomers and the solvent accessible residues of BSA. Such novel chiral recognition approach may have a great potential in the development of chiral sensing technology preferable to L-amino acid without using an enantioresolvable separation column, which can be used in diverse sensing systems for various chiral compounds and is of great importance in life science and biomaterial science. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel.: 86-519-86330253. Fax: 86-519-86330167. 21 ACS Paragon Plus Environment


Page 22 of 29

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This research was supported financially by National Natural Science Foundation of China (21804013, 31670950), Natural Science Foundation of Jiangsu Province (BK20171194), Advanced

Catalysis

and

Green

Manufacturing

Collaborative

Innovation

Center

(ACGM2016-06-27) and by Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). ASSOCIATED CONTENT Supporting Information Additional figures as noted in the text, the combination of Trp isomers with the fifteen segments of solvent accessible residues, and water contact angle measurements on different samples. This material is available free of charge via the Internet at http://pubs.acs.org.

REFERENCES (1) Rubenstein, E.; Bonner, W. A.; Noyes, H. P.; Brown, G. S. Supernovae and Life. Nature 1983, 306, 118–118. (2) Biedermann, F.; Nau, W. M. Noncovalent Chirality Sensing Ensembles for the Detection and Reaction Monitoring of Amino Acids, Peptides, and Aromatic Drugs. Angew. Chem. Int. Ed. 2014, 53, 5694–5699. 22 ACS Paragon Plus Environment

Page 23 of 29 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


(3) Han, C. P.; Hou, X.; Zhang, H. C.; Guo, W.; Li, H. B.; Jiang, L. Enantioselective Recognition in Biomimetic Single Artificial Nanochannels. J. Am. Chem. Soc. 2011, 133, 7644−7647. (4) Kempe, M.; Mosbach, K. Separation of Amino Acids, Peptides and Proteins on Molecularly Imprinted Stationary Phases. J. Chromatogr. A 1995, 691, 317–323. (5) Whitcombe, M. J.; Vulfson, E. N. Imprinted Polymers. Adv. Mater. 2001, 13, 467–478. (6) Gassmann, E.; Kuo, J. E.; Zare, R. N. Electrokinetic Separation of Chiral Compounds. Science 1985, 230, 813–814. (7) Tang, M. L.; Zhang, J.; Zhuang, S. L.; Liu, W. P. Development of Chiral Stationary Phases for High-Performance Liquid Chromatographic Separation. Trends Anal. Chem. 2012, 39, 180–194. (8) Schneider, H. J.; Agrawal, P.; Yatsimirsky, A. K. Supramolecular Complexations of Natural Products. Chem. Soc. Rev. 2013, 42, 6777–6800. (9) Kimmel, D. W.; LeBlanc, G.; Meschievitz, M. E.; Cliffel, D. E. Electrochemical Sensors and Biosensors. Anal. Chem. 2012, 84, 685–707. (10) Tao, Y. X.; Gu, X. G.; Deng, L. H.; Qin, Y.; Xue, H. G.; Kong, Y. Chiral Recognition of D-Tryptophan by Confining High-Energy Water Molecules Inside the Cavity of Copper-Modified β-Cyclodextrin. J. Phys. Chem. C 2015, 119, 8183−8190. (11) Tao, Y. X.; Dai, J. Y.; Kong, Y.; Sha, Y. Temperature-Sensitive Electrochemical Recognition of Tryptophan Enantiomers Based on β-Cyclodextrin Self-Assembled on Poly(L-Glutamic Acid). Anal. Chem. 2014, 86, 2633–2639. (12) Trojanowicz, M. Enantioselective Electrochemical Sensors and Biosensors: A 23 ACS Paragon Plus Environment


Mini-Review. Electrochem. Commun. 2014, 38, 47−52. (13) Yu, Y.; Tao, Y. X.; Yang, B. Z.; Wu, D. T.; Qin, Y.; Kong, Y. Smart Chiral Sensing Platform with Alterable Enantioselectivity. Anal. Chem. 2017, 89, 12930–12937. (14) Guo, L. L.; Yang, B. Z.; Wu, D. T.; Tao, Y. X.; Kong, Y. Chiral Sensing Platform Based on the Self-Assemblies of Diphenylalanine and Oxalic Acid. Anal. Chem. 2018, 90, 5451–5458. (15) Li, Z. Y.; Xu, H.; Wu, D. T.; Zhang, J.; Liu, X. R.; Gao, S. M.; Kong, Y. Electrochemical Chiral Recognition of Tryptophan Isomers Based on Nonionic Surfactant-Assisted Molecular Imprinting Sol−Gel Silica. ACS Appl. Mater. Interfaces 2019, 11, 2840−2848. (16) Narayanan, S. R. Immobilized Proteins as Chromatographic Supports for Chiral Resolution. J. Pharm. Biomed. Anal. 1992, 10, 251–262. (17) Hermansson, J. Enantiomeric Separation of Drugs and Related Compounds Based on Their Interaction with α1-Acid Glycoprotein. TrAC, Trends Anal. Chem. 1989, 8, 251–259.


Page 24 of 29

Page 25 of 29 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


(18) Nosrati, H.; Salehiabar, M.; Manjili, H. K.; Danafar, H.; Davaran, S. Preparation of Magnetic Albumin Nanoparticles via a Simple and One-Pot Desolvation and Co-Precipitation Method for Medical and Pharmaceutical Applications. Int. J. Biol. Macromol. 2018, 108, 909–915. (19) Liu, Y.; Chen, M.; Wang, S.; Lin, J.; Cai, L.; Song, L. New Insight into the Stereoselective Interactions of Quinine and Quinidine, with Bovine Serum Albumin. J. Mol. Recognit. 2014, 27, 239–249. (20) Majorek, K. A.; Porebski, P. J.; Dayal, A.; Zimmerman, M. D.; Jablonska, K.; Stewart, A. J.; Chruszcz, M.; Minor, W. Structural and Immunologic Characterization of Bovine, Horse, and Rabbit Serum Albumins. Mol. Immunol. 2012, 52, 174–182. (21) Hu, X.; Liu, Y.; Jiang, Y. H.; Meng, M. J.; Liu, Z. C.; Ni, L.; Wu, W. F. Construction and Comparison of BSA-Stabilized Functionalized GQD Composite Fluorescent Probes for Selective Trypsin Detection. New J. Chem. 2018, 42, 17718–17724. (22) Krishnan, S. K.; Singh, E.; Singh, P.; Meyyappan, M.; Nalwa, H. S. A Review on Graphene-Based Nanocomposites for Electrochemical and Fluorescent Biosensors. RSC Adv. 2019, 9, 8778–8881. (23) Gogoi, A.; Mazumder, N.; Konwer, S.; Ranawat, H.; Chen, N. T.; Zhuo, G. Y. Enantiomeric Recognition and Separation by Chiral Nanoparticles. Molecules 2019, 24, 1007.



(24) Zhu, S. J.; Tang, S. J.; Zhang, J. H.; Yang, B. Control the Size and Surface Chemistry of Graphene for the Rising Fluorescent Materials. Chem. Commun. 2012, 48, 4527–4539. (25) Shen, J. H.; Zhu, Y. H.; Yang, X. L.; Li, C. Z. Graphene Quantum Dots: Emergent Nanolights for Bioimaging, Sensors, Catalysis and Photovoltaic Devices. Chem. Commun. 2012, 48, 3686–3699. (26) Dong, Y.; Shao, J.; Chen, C.; Li, H.; Wang, R.; Chi, Y.; Lin, X.; Chen, G. Blue Luminescent Graphene Quantum Dots and Graphene Oxide Prepared by Tuning the Carbonization Degree of Citric Acid. Carbon 2012, 50, 4738–4743. (27) Ou, J.; Zhu, Y. H.; Kong, Y.; Ma, J. F. Graphene Quantum Dots/β-Cyclodextrin Nanocomposites: A Novel Electrochemical Chiral Interface for Tryptophan Isomer Recognition. Electrochem. Commun. 2015, 60, 60−63. (28) Tanaka, Y.; Terabe, S. Recent Advances in Enantiomer Separations by Affinity Capillary Electrophoresis Using Proteins and Peptides. J. Biochem. Biophys. Methods 2001, 48, 103−116. (29) Qian, Z. S.; Shan, X. Y.; Chai, L. J.; Ma, J. J.; Chen, J. R.; Feng, H. DNA Nanosensor Based on Biocompatible Graphene Quantum Dots and Carbon Nanotubes. Biosens. Bioelectron. 2014, 60, 64–70. (30) Mandeville, J. S.; Tajmir-Riahi, H. A. Complexes of Dendrimers with Bovine Serum Albumin. Biomacromolecules 2010, 11, 465−472. (31) Das, S.; Islam, M. M.; Jana, G. C.; Patra, A.; Jha, P. K.; Hossain, M. Molecular Binding of Toxic Phenothiazinium Derivatives, Azures to Bovine Serum Albumin: A 26 ACS Paragon Plus Environment

Page 26 of 29

Page 27 of 29 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


Comparative Spectroscopic, Calorimetric, and in Silico Study. J. Mol. Recognit. 2017, 30, e2609. (32) Ou, J.; Tao, Y. X.; Xue, J. J.; Kong, Y.; Dai, J. Y.; Deng, L. H. Electrochemical Enantiorecognition of Tryptophan Enantiomers Based on Graphene Quantum Dots–Chitosan Composite Film. Electrochem. Commun. 2015, 57, 5−9. (33) Shen, J. H.; Zhu, Y. H.; Chen, C.; Yang, X. L.; Li, C. Z. Facile Preparation and Upconversion Luminescence of Graphene Quantum Dots. Chem. Commun. 2011, 47, 2580−2582. (34) Sudha, A.; Srinivasan, P.; Thamilarasan, V.; Sengottuvelan, N. Exploring the Binding Mechanism of 5-Hydroxy-3′,4′,7-Trimethoxyflavone with Bovine Serum Albumin: Spectroscopic and Computational Approach. Spectrochim. Acta A 2016, 157, 170−181. (35) Kamat, B. P. Study of the Interaction Between Fluoroquinolones and Bovine Serum Albumin. J. Pharm. Biomed. Anal. 2005, 39, 1046−1050. (36) Wang, X. D.; Chen, L. J.; Su, X. R.; Ai, S. Y. Electrochemical Immunosensor with Graphene

Quantum

Dots

and

Apoferritin-Encapsulated

Cu

Nanoparticles

Double-Assisted Signal Amplification for Detection of Avian Leukosis Virus Subgroup J. Biosens. Bioelectron. 2013, 47, 171−177. (37) Huang, K. J.; Xu, C. X.; Xie, W. Z.; Wang, W. Electrochemical Behavior and Voltammetric Determination of Tryptophan Based on 4-Aminobenzoic Acid Polymer Film Modified Glassy Carbon Electrode. Colloids Surf., B 2009, 74, 167–171. (38) Gu, X. G.; Tao, Y. X.; Pan, Y.; Deng, L. H.; Bao, L. P.; Kong, Y. DNA-Inspired Electrochemical Recognition of Tryptophan Isomers by Electrodeposited Chitosan and 27 ACS Paragon Plus Environment


Sulfonated Chitosan. Anal. Chem. 2015, 87, 9481−9486. (39) DeLano, W. L. The PyMOL Molecular Graphics System 2002. (40) Dreuw, A.; Head-Gordon, M. Single-Reference ab Initio Methods for the Calculation of Excited States of Large Molecules. Chem. Rev. 2005, 105, 4009−4037. (41) Zhao, Y.; Truhlar, D. G. Density Functionals with Broad Applicability in Chemistry. Acc. Chem. Res. 2008, 41, 157−167. (42) Desiraju, G.; Steiner, T. The weak hydrogen bond in structural chemistry and biology, Oxford University Press: Oxford, 1999.


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For TOC only


Covalent Functionalization of Bovine Serum Albumin with Graphene

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