Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 2015 Supporting Information A new ICT and CHEF based visible light excitable fluorescent probe easily detects in vivo Zn 2+ Krishnendu Aich, [a] Shyamaprosad Goswami,* [a] Sangita Das [a] and Chitrangada Das Mukhopadhyay [b] a Department of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur, Howrah-711 103, India. Fax: +91 33 2668 2916; Tel: +91 33 2668 2961-3; E-mail: spgoswamical@yahoo.com b Department of of Centre for Healthcare Science & Technology, Indian Institute of Engineering Science and Technology, Shibpur, Howrah-711 103, India. CONTENTS 1. Determination of detection limit.. 2. Determination of association constant 3. Job plot.. 4. Competition Study. 5. Determination of Quantum yield. 6. ph study. 7. 1 H NMR spectrum of BQ 8. 13 C NMR spectrum of BQ.. 9. Mass spectrum (HRMS) of BQ. 10. MS spectrum of Zn 2+ complex of BQ....
1. Determination of detection limit: The detection limit was calculated based on the absorption and fluorescence titration. To determine the S/N ratio, the emission intensity of BQ without Zn 2+ was measured by 10 times and the standard deviation of blank measurements was determined. The detection limit is then calculated with the following equation: DL = K* Sb 1 /S Where K = 2 or 3 (we take 3 in this case); Sb 1 is the standard deviation of the blank solution; S is the slope of the calibration curve. For Zn 2+ : From the graph we get slope = 1.138 10 11, and Sb 1 value is 892.32. Thus using the formula we get the Detection Limit = 2.35 10-8 M i.e. BQ can detect Zn 2+ in this minimum concentration by fluorescence techniques. Intensity at 475 nm 1.2x10 6 8.0x10 5 4.0x10 5 (a) Intensity at 475 nm 1.0x10 6 8.0x10 5 6.0x10 5 4.0x10 5 2.0x10 5 Equation y = a + b* Adj. R-Squar 0.99045 Value Standard Err B Intercept 114910.9680 19885.66983 B Slope 1.13815E11 4.55782E9 (b) 0.0 0.0 4.0x10-6 8.0x10-6 1.2x10-5 0.0 0.0 2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 Figure S1: Emission of BQ at 475 nm depending on the concentration of Zn 2+
From the graph we get slope = 28469.227, and Sb 1 value is 0.00504, Thus using the formula we get the Detection Limit = 5.31 10-7 M i.e. BQ can detect Zn 2+ in this minimum concentration by UV/vis techniques. 0.4 0.3 0.30 0.25 Equation y = a + b Adj. R-Squa 0.98814 Value Standard Err B Intercept 0.06005 0.00707 B Slope 28469.227 1392.94412 A405/A282 0.2 0.1 A405/A282 0.20 0.15 0.10 0.0 0.0 4.0x10-6 8.0x10-6 1.2x10-5 0.05 0.0 3.0x10-6 6.0x10-6 9.0x10-6 Figure S2: Absorbance ratio of BQ at (A 405 /A 282 ) depending on the concentration of Zn 2+ Absorbance at 405 nm 0.10 0.08 0.06 0.04 0.02 0.00 0.0 4.0x10-6 8.0x10-6 1.2x10-5 Absorbance at 405 nm 0.08 0.06 0.04 0.02 0.00 Equation y = a + b*x 0.9641 Adj. R-Square Value Standard Error B Intercept 0.00612 0.00479 B Slope 12604.93533 1210.54925 0.0 3.0x10-6 6.0x10-6 Figure S3: Absorbance BQ at (A 405 ) depending on the concentration of Zn 2+
2. Determination of Association Constant (K a ): By UV-vis method: Association constant was calculated according to the Benesi-Hildebrand equation. K a was calculated following the equation stated below. 1/(A-A o ) = 1/{K(A max A o ) [M x+ ] n } + 1/[A max -A o ] Here A o is the absorbance of receptor in the absence of guest, A is the absorbance recorded in the presence of added guest, A max is absorbance in presence of added [M x+ ] max and K a is the association constant, where [M X+ ] is [Zn 2+ ]. The association constant (K a ) could be determined from the slope of the straight line of the plot of 1/(A-A o ) against 1/[Zn 2+ ] and is found to be 5.77 10 5 M -1. 50 40 Equation y = a + b* Adj. R-Square 0.97098 Value Standard Error B Intercept 10.69005 1.15566 B Slope 1.85198E- 1.30379E-6 1/(A-A0) 30 20 10 0.0 5.0x10 5 1.0x10 6 1.5x10 6 2.0x10 6 1/[Zn 2+ ] Figure S4: Benesi Hildebrand plot from UV/vis titration data of receptor (10 µm) with Zn 2+ concentration By fluorescence method: The binding constant value of Zn 2+ with receptor has been determined from the emission intensity data following the modified Benesi Hildebrand equation, 1/ I = 1/ I max +(1/K a [C])(1/ I max ). Here I = I I min and I max = I max I min, where I min, I, and I max are the emission intensities of receptor considered in the absence of Zn 2+, at an intermediate Zn 2+
concentration, and at a concentration of complete saturation where K is the binding constant and [C] is the Zn 2+ concentration respectively. From the plot of [1 / (I I min )] against [C] -1 for receptor, the value of K has been determined from the slope. The association constant (K a ) as determined by fluorescence titration method for the receptor with Zn 2+ is found to be 2.48 10 5 M -1. 5.0x10-6 4.0x10-6 Equation y = a + b*x Adj. R-Square 0.9876 Value Standard Error B Intercept 5.47653E-7 8.0673E-8 B Slope 2.20368E-12 9.32526E-14 1/(I-I min ) 3.0x10-6 2.0x10-6 1.0x10-6 0.0 5.0x10 5 1.0x10 6 1.5x10 6 2.0x10 6 1/[Zn 2+ ] Figure S5: Benesi Hildebrand plot from fluorescence titration data of receptor (10 µm) with Zn 2+. 3. General procedure for drawing Job s plot by fluorescence method: Stock solution of same concentration of sensor and Zn 2+ was prepared in the order of 10 μm in [CH 3 OH/ H 2 O, 1/9, v/v] (at 25 C) at ph 7.4 in HEPES buffer. The absorbance spectrum in each case with different host guest ratio but equal in volume was recorded. Job s plots were drawn by plotting ΔI.X host vs X host (ΔI = change of absorbance of the absorption spectrum at 405 nm during titration and X host is the mole fraction of the host in each case, respectively).
0.08 0.06 I.Xh 0.04 0.02 0.00 0.0 0.2 0.4 0.6 0.8 1.0 Xh Figure S6: Job s plot diagram of receptor for Zn 2+ (where X h is the mole fraction of the host and ΔI indicates the change of absorbance at 405 nm). 4. Competition study Intensity at 475 nm 1.0x10 6 8.0x10 5 6.0x10 5 4.0x10 5 2.0x10 5 0.0 BQ Na + K + Mg 2+ Cu 2+ Mn 2+ Fe 2+ Fe 3+ Co 2+ Ni 2+ Cd 2+ Cr 3+ Hg 2+ Figure S7: Competition study using Fluorescence method, after addition of different analytes (30 μm) in the solution of BQ (10 μm) in presence of Zn 2+ (20 μm).
5. Determination of fluorescence Quantum Yields (Φ) of BQ and its complex with Zn 2+ ion: For measurement of the quantum yields of BQ and its complex with Zn 2+, we recorded the absorbance of the compounds in methanol solution. The emission spectra were recorded using the maximal excitation wavelengths, and the integrated areas of the fluorescencecorrected spectra were measured. The quantum yields were then calculated by comparison with fluorescein (Φs = 0.97 in basic ethanol) as reference using the following equation: Φx = Φs ( Ix Is) ( Ax) As ( nx ns) 2 Where, x & s indicate the unknown and standard solution respectively, Φ is the quantum yield, I is the integrated area under the fluorescence spectra, A is the absorbance and n is the refractive index of the solvent. We calculated the quantum yield of BQ and BQ-Zn 2+ using the above equation and the value is 0.02 and 0.22 respectively. 6. ph study Intensity at 475 nm 1.2x10 6 9.0x10 5 6.0x10 5 3.0x10 5 0.0 (a) 2 4 6 8 10 12 ph Intensity at 475 nm 1.00x10 6 7.50x10 5 5.00x10 5 2.50x10 5 0.00 (b) 2 4 6 8 10 Wavelength (nm)
Figure S8: Fluorescence response of (a) BQ and (b) BQ-Zn 2+ at 475 nm (10 µm) as a function of ph in CH 3 OH/ H 2 O (1/ 9, v/v), ph is adjusted by using aqueous solutions of 1 M HCl or 1 M NaOH Comparison of present probe with the existing probes: Table S1: The comparison of the present probe with recently reported probes for Zn 2+ have been outlined in this table. Fluorophore used Type of response Quinoline-pyridine Colorimetric, Quinoline-pyridine Colorimetric, Pentaquinone Colorimetric, Fluorometric Pyridine-hydrazone Colorimetric, Detection limit 6.6 10-8 M 4.9 10-8 M Living cell imaging Reference No Dalton Trans., 2013, 42, 15514 Yes Dalton Trans., 2014, 43, 706 713 3.5 10-9 No Dalton Trans., Ml -1 2013, 42, 975 69 ppb Yes Org. Biomol. Chem., 2014, 12, 4975. benzothiazole benzoxazole 8-aminoquinoline benzaldehyde Quinoline Colorimetric, Colorimetric, Colorimetric, Colorimetric, Colorimetric, 67 μm Yes Dalton Trans., 2015, 44, 2097 2102 1.63 10-8 Yes Chem.Commun., M 2014, 50, 7514. 10-7 M No New J. Chem., 2014, 38, 1802. 76 μm Yes Dalton Trans., 2.35 10-8 M Yes 2013, 42, 16569. Present work
7. 1 H NMR spectrum of BQ Figure S9: 1 H NMR (400 MHz) spectrum of BQ in DMSO-d 6. 8. 13 C NMR of BQ
Figure S10: 13 C NMR (125 Hz) spectrum of BQ in DMSO-d 6. 9. HRMS of BQ Figure S11: HRMS of BQ.
Figure S11a: HRMS (expansion) of BQ. 10. HRMS of BQ-Zn 2+ complex: Figure 12: HRMS of BQ-Zn 2+ complex