Supporting information
Studies on the aggregation induced synchronous emission of1,8-naphthalimide derivativeto casein and its analytic application
Yang Suna,*, Xuhua Liangb, Yingyong Zhaoc, Jun Fanb,*
a Department of Chemistry & Chemical Engineering, Xi’an University of Arts and Science, No. 168 Taibai South Road, Xi’an, Shaanxi 710065, China
b School of Chemical Engineering, Northwest University, No.229 Taibai North Road, Xi’an, Shaanxi 710069, China
c College of Life Sciences, Northwest University, No.229 Taibai North Road, Xi’an, Shaanxi 710069,China
* Corresponding author. Tel.: +86 29 88305252;
E-mail address: (Y. Sun), (J. Fan)
Content list
1. Experimental details…………………………………………………………………….…..S2
1.1 Materials……………………………………………………………………………….…..S2
1.2 Apparatus……………………………………………………………………………….…S2
1.3 Methods…………………………………………………………………….………….…..S2
1.3.1Synthesis of probe 1…………………………………………………….………………..S2
1.3.2Spectroscopic measurements………...…………………...…………….………………..S3
1.3.3 Synchronous fluorescence spectrafor system of 1 and human serum albumin ....…..….S3
1.3.4Accuracy and precision……………………………………………………...……….…..S4
1.3.5Casein quantification in milk samples………...…… ……………………..…………S6
1. Experiment details
1.1. Materials
All the solvents were of analytic grade. 4-bromo-1,8-naphthalic anhydride and1-(2-pyridyl)piperazine were purchased from Aldrich. Casein (90%), lactoglobulin, whey and HSA were purchased from Sigma without further treatment. Casein stock solution was prepared with the concentration of 10.0 mg mL–1. Some drops of 0.1 M NaOH solution were added to help solving casein, and 0.1 M HCl was applied for adjusting the pH of casein solution to pH 7.0. We diluted the casein stock solution with Tris–HCl buffer, and the dispersions were stored at 4 °C overnight to allow complete hydration. Stock solution of 1(1.5 × 10–5 M) was prepared by dissolving the appropriate amount of 1in Milli-Q water and kept in darkness.
Figure S1. Synthetic approachof probe 1
1.2. Apparatus
EQUINOX55 Fourier transformed infrared spectrometry (Bruker Company, Germany). AVANCF300 MHz digital superconducting NMR Fourier (Bruker Company, Germany). X-5 microscopic melting point apparatus (Tech Instrument Co., Ltd. Beijing, China).The fluorescence measurements were carried out on an F-7000 fluorescence spectrometer (Hitachi, Japan) equipped with a 150W Xenon lamp. A 1.0 cm quartz cell was used for measurements. Elemental analysis data were obtained on a Perkin–Elmer 240c instrument.
1.3 Method
1.3.1.Synthesis of probe 1
The intermediate 2 was prepared according to the reported procedures, and its structure was characterized by chemical and spectroscopic methods (1H NMR, IR) and compared with the data reported in the literature [S1]. A mixture of 2 (0.27 g), sodium hydroxide solid particles (0.13 g), cetyltrimethylammonium bromide (CTAB, 0.13 g) and 1-(2-pyridyl)piperazine (1 mL) were dissolved in 10 mL of 2-methoxyethanol. The mixture was stirred and heated at 60 °C for 6 h, and the reaction was monitored by TLC (using CH2Cl2 as eluent). After cooling, the solvent was evaporated and the product was extracted with a 0.5 M solution of cloroform/EtOH (9:1) to give a yellow precipitate (75 %). The synthetic route was listed in Fig. S1 (Supporting Information). m.p. 324–328oC.; IR (cm-1): 2831 (νCH); 1734 (νC=O); 1692(νas naphthalimide N-C=O); 1657 (νa naphthalimide N-C=O).1H NMR (400 MHz, CDCl3) δ: 7.98~7.82 (d, J =7.1 Hz, 4H), 7.45~7.21 (t, J = 8.8 Hz, 3H), 6.76~6.63 (d, J = 8.4 Hz, 2H), 3.51~3.20 (t, J =7.2Hz, 12H), 3.56~3.34 (s, 6H).
[S1] Dai, H. L.; Xu,H. Bioor. Med. Chem. Lett., 2011,21, 5141–5144.
1.3.2. Spectroscopic measurements
The effect of various concentration of 1on the emission spectra of casein was collected with the excitation wavelength at 280 nm.Synchronous fluorescence spectra of casein with various concentrations of 1 were obtained from 200 to 450 nm (Δλ = 15 nm and Δλ = 60 nm). The effect of Fe3+ or Cu2+ion on the excitationspectra of1was collected with the emission wavelength at 550 nm.
1.3.3. The Synchronous fluorescence spectra for system of 1 and human serum albumin
In order to verify whetherthefluorescenceenhancement of 1caused by casein structure, we studied the synchronous fluorescence spectrafor system of1-HSA (Fig. S2). As seen from Fig. S1, the trend of decreasing intensity and emission shift are similar to casein, however, the fluorescencefailed to be turned on by casein in the presence of HSA. This suggests that the enhanced fluorescence of 1results from the micelle and structure of casein.
Figure S2 Synchronous fluorescence spectrafor system of 1-HSA
Table S1 Compared with other methods in detectionlimit
Fluorescence medium / Protein / Detectionlimit (ng mL–1) / Ref.Biosensor immunoassay / αS1-CN / 870 / S2
RPHPLC-MS / β-Casein / 210 / S3
ELISA / Casein / 300 / S4
Immunochromatographic / κ-Casein / 300 / S5
This method / Casein / 4.5/6.7
[S2] Stéphanie M.R., Didier D., Philippe D. J. Dairy Res., 2005, 72, 57-64.
[S3] Feligini M., Bonizzi I., Buffoni J.N., Cosenza G., Ramunno L. J. Agric. Food Chem. 2009, 57 (7), 2988–2992.
[S4] Patrick W., Hans S., Angelika P. J. Agric. Food Chem. 2009, 57 (18), 8399–8405.
[S5] Hernández C.M., Muñoz M., Daury C., Weymuth H., Kemmers-Voncken A.E.M., Corbatón V., Toribio T., Bremer M.G.E.G. Int. Dairy J. 2009, 19(4), 205–208.
1.3.4. Accuracy and precision
Table S2 showed theaccuracy and precision of proposed method for detecting casein. The precisions were measured below 7.0 % in the relative recoveryrange between 97.7% and 101.3%. Our results demonstrated that the proposed method is precise and accurate, especially for the detection of casein at low levels.
Table S2 Summary of precision and accuracy of detecting casein by proposed method (n = 6 assays, six time replicates per assay)
Sample con.(μg mL–1) / Added con.
(μg mL–1) / Found con.
(mean ± SD, μg mL–1) / Recovery (%) / RSD
(%)
0 / 0.1 / 0.10 ± 0.02 / 100 ± 0.02 / 3.2
0.1 / 0.2 / 0.30 ± 0.03 / 100 ± 0.03 / 3.1
0.5 / 0.3 / 0.81 ± 0.03 / 101.2 ± 0.2 / 2.2
1 / 0.5 / 1.52± 0.18 / 101.3 ± 0.15 / 3.1
1.5 / 0.7 / 2.16± 0.20 / 98.2± 0.62 / 4.3
2 / 0.9 / 3.09± 0.23 / 106.6± 0.59 / 3.9
2.5 / 1.1 / 3.53± 0.22 / 98.1±0.52 / 2.2
3 / 1.3 / 4.24± 0.21 / 98.6± 0.47 / 4.1
Table S3 Effect of interfering substances
Foreign substance / Concentration(μM) / Relative error (%) / Foreign substance / Concentration
(μM) / Relative error (%)
Na+ / 30 / 1.01 / Lactose / 10 / 3.35
K+ / 30 / 1.23 / Chitosan / 10 / 3.21
Ca2+ / 5 / 1.31 / Trehalose / 10 / 3.10
Mg2+ / 5 / 2.14 / Sucrose / 10 / 1.15
Ni2+ / 5 / 1.78 / Urea / 12 / –4.17
Pb2+ / 5 / 2.19 / Melamine / 12 / –4.22
Zn2+ / 5 / –2.58 / Whey / 10 / 4.43
Ag+ / 5 / –1.34 / HSA / 10 / 2.21
Cr3+ / 5 / 2.56 / BSA / 10 / 2.14
Mn2+ / 5 / 2.40 / Tryptophane / 10 / 2.85
Cu2+ / 5 / –20.13 / Tyrosine / 10 / 1.92
Fe3+ / 5 / –18.29 / SDS / 15 / 4.56
Lecithin / 10 / 4.12 / CTAB / 15 / 4.12
Glucose / 10 / 2.01 / Tween / 15 / 4.36
Figure S3Mechanism of probe 1 binding with metal ions.
1.3.5. Casein quantification in milk powder samples
1) 50 mg milk powder was dissolved in 10 ml Tris-HCl buffer (pH 7.4).
2) Milk solution was centrifuged for 10 minutes at 4000 rpm and the upper layer of fatty acid was peeled off.
3) HCl (0.1 M) was added to adjust pH to 4.7, casein would be deposited. The solution was centrifuged for 10 min at 10000 rpm to get the precipitate.
4) The precipitate was washed with ethanol for 2 times to remove fatty acid, and then the precipitate was washed with buffer for 2 times. A few drops of 0.1 M NaOH were added to help solving the casein precipitate and the final volume for sample solution was 3 mL. Finally, the pH was adjusted to pH 7.0 with 0.1MHCl.10 μL casein sample was added to a quartz cell with the 1 solution, and the fluorescence spectra were collected with the wavelength interval (Δλ)is 15or 60 nm.
Table S4. The milk powder samples information
Sample / Remarks / Manufacturer / Company1 / Full milk First stage / Wheth / New Zealand
2 / Full milk Second stage / Nestlé / Switzerland
3 / Full milk Third stage / MeadJohnson / USA
4 / Full milk for female / Abbott / USA
5 / Full milk for elderly / Dumex / France
6 / Full milk for pregnant / Yili / China
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