Electronic Supplementary Information (ESI) Silk fibroin as a platform for dual sensing of vitamin B12 using photoluminescence and electrical techniques Sudesna Chakravarty1#, Bedanta Gogoi1#, Biman B. Mandal2, Nandana Bhardwaj3,* and Neelotpal Sen Sarma1,* 1
Advanced Materials Laboratory, Physical Sciences Division, Institute of Advanced Study in Science & Technology, Guwahati-781035, India 2
Tissue Engineering and Biomaterials Laboratory, Department of Bioscience and Bioengineering, Indian Institute of Technology Guwahati, Guwahati-781039, India
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Seri-Biotechnology Laboratory, Life Sciences Division, Institute of Advanced Study in Science & Technology, Guwahati- 781035, India
*Corresponding authors: Dr. Neelotpal Sen Sarma Tel: +91 361 2912073 Fax: +91 361 2279909 E-mail:
[email protected] Dr. Nandana Bhardwaj Tel: 0361-2270095 Fax: 0361-2740659 E-mail:
[email protected],
[email protected] #
These authors contributed equally
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1. Materials B. mori silk cocoons were procured from Mangaldoi silk farm, Guwahati. Vitamin B12 (B12) (SRL), ethanol (Merck), methanol (Merck), sodium carbonate (Merck), lithium bromide (Sigma), polydimethylsiloxane (PDMS) [Sigma], cytochrome C (SRL), bovine serum albumin (SRL), retinol (Sigma), tryptophan (SRL), phenyl alanine (SRL), L-DOPA (SRL), glycine (SRL), cysteine (SRL) and aspartic acid (RL). All reagents were used as received without any further purification. 2. Extraction of silk-fibroin protein Silk cocoons were cut into pieces and degummed in 0.02 M Na2CO3 solution under boiling conditions for 30 mins to remove sericin. Fibers were further washed with deionized water followed by drying overnight at 37 oC. Purified and dried fibroin fibers were dissolved in 9.3 M LiBr and further dialyzed against Milli Q-treated water using a 12 kDa molecular weight cut-off cellulose dialysis membrane with frequent change of water at a regular time interval for 48 hrs. A solution of BMSF was prepared by adjusting the composition to 1 % w/v. The concentration of SF was determined gravimetrically and adjusted to the prescribed concentration for each experiment by diluting if necessary. 0.5 % SF solution was utilized for experiments by diluting the stock solution. 3. Fabrication of BMSF thin films BMSF aqueous solution based thin films were prepared using earlier established protocol for characterization studies (Rockwood et al., 2011). The fabricated films were treated with ethanol (70 % for 30 minutes) in order to induce β-sheets formation and providing subsequent stability. This was followed with repetitive washing (4-6 times) with phosphate buffered saline (PBS, pH-7.4) in order to remove alcohol. Finally, the films were vacuum dried at 45 oC for about an hour and used for characterization experiments. 4. Physico-chemical characterization 4.1. Field emission scanning electron microscopy (FESEM)
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The surface morphology of the micro patterned films was studied with a SIGMA VP (ZEISS) FESEM at an accelerating voltage of 5 kV. Briefly, thin films of the samples were sputter coated with gold for 6 min before analysis. 4.2 Conformational analysis FT-IR studies were carried out in Nicolet 6700 (Thermo Fischer) FT-IR with an Attenuated Total Reflectance (ATR) attachment. Thin films of dimension 1 cm × 1 cm were used for analysis 4.3 Dynamic Light Scattering Study (DLS) Room temperature zeta potential of the solutions was measured in Malvern NanoZS90 in a glass cuvette with square aperture with zeta dip cell electrode. Hydrodynamic radius of the particles were also measured in the glass cuvette with square aperture. 4.4 X-Ray Diffraction Study (XRD) The crystalline properties of water annealed BMSF films were analysed using a Bruker D 8 Advance X-ray diffractometer with Cu Kα radiation, λ=0.154 nm, and a Ni filter. The tube current was maintained at 40 mA with a tube voltage of 40 kV. The angular range was 2θ =10-90ο. 4.5 Thermogravimetric Analysis (TGA) TGA (Perkin Elmer TGA 4000) was used to measure changes in weight of the treated and untreated BMSF films with increasing temperature. TGA curves were obtained under a non-oxidative environment of nitrogen with a gas flow of 20 ml min-1 and at a heating rate of 5 oC/min. 4.6 UV-Vis spectral Study The electronic transitions associated in the present context were observed by recording UV-Vis spectra at room temperature in Shimadzu UV1601PC spectrophotometer. 4.7 Photoluminescence study (PL) The PL studies were done using Agilent Varian Cary Eclipse spectrophotometer with a halogen lamp as the excitation source, at a scan speed of 240 nms-1. The excitation and emission slit widths were maintained at 5 nm and the scan rate was kept constant for all the experiments. The detector voltage
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was maintained at 550 V. Quartz cells (4×1×1 cm) with high vacuum Teflon stopcocks were used for spectral measurements. 4.8 Fluorescence microscopy Fluorescent images of silk films were obtained using a fluorescence microscope (Leica DMI 3000 B) under FITC and rhodamine monochromatic light. 4.9 Atomic force microscope (AFM) measurements The AFM measurements of the micro patterned films were performed using NTRGRA Prima NTMDT AFM system. The AFM images were collected in contact mode measurements using NT-MDT NSG10 AFM tips. 4.10 Electrical measurements The electrical measurements were performed using HIOKI 3532-50, LCR Hi-TESTER. 5. Fabrication and physico-chemical characterization of silk fibroin films Regenerated silk fibroin protein surface morphology in the form of micro patterned films (MF) was assessed by FESEM. Herein, the MF were treated with ethanol for induction of β-sheets and subsequent stability. Alcohol treatment leads to a rougher and globular structure (Fig. S1A) (Mandal and Kundu, 2008). FESEM images indicated the distance between the grids of the micro patterned film is about 2.6 micro meter. Furthermore, conformational changes were determined using FTIR and XRD. Fig. S1B depicts the FTIR spectra of silk fibroin solution and alcohol treated films, which indicated characteristic absorption peaks assigned to the representative amide bonds-amide I, amide II and amide III. The characteristic amide I band, which represents C=O stretching lies in the range of 1600-1700 cm-1. In contrast, amide II and amide III represents N-H bending and C-N stretching are mainly confined to 1520-1540 cm-1 and 1220-1300 cm-1, respectively (Bhardwaj et al., 2011; Bhardwaj et al., 2014). In this study, silk fibroin solution indicated peaks at 1658 cm-1, 1541 cm-1, and 1241 cm-1, which signifies the α-helical conformation and amorphous structure. However, alcohol treated films indicated characteristics amide peaks at 1625 cm-1, 1520 cm-1 and 1232 cm-1, respectively, which signifies the
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typical β-sheet crystalline silk-II structure (Bhardwaj et al. 2014). Silk I and silk II represents two main types of crystalline structures for silk. Silk I structure represents the regenerated structure prior to crystallization. In contrast, silk II comprises represents the spun silk state, which mainly comprises of a β-sheet secondary structure (Bhardwaj et al. 2011). Furthermore, in order to conform the secondary conformation, XRD analysis was performed [Fig. S1C]. Herein, two broad peaks were observed at around 16.80 and 20.20. These peaks signify silk-II β-sheet secondary structure (Bhardwaj et al. 2015). These results demonstrate stable and crystalline structure of silk fibroin films in agreement with the earlier reports.
Fig. S1. (A) FESEM micrograph of micro patterned MF, (B) FT-IR spectra of B. mori silk fibroin regenerated solution (a) and alcohol treated MF (b), (C) XRD spectra of alcohol treated MF and, (D) TGA thermogram of MF.
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The thermal stability of the MF were further determined by thermogravimetric analysis (TGA) (Fig. S1D). TGA analysis showed weight loss pattern and indicated 2 step breakdown. The first weight loss was observed around 101-104 0C, while second weight loss at around 273 0C. The first and second endothermic peaks signify the loss of moisture and decomposition of protein molecule, respectively (Bhardwaj et al. 2015) . The results corroborated with the earlier results and demonstrated the thermally stable structure. 6. Photoluminescence study 6.1 Stern-Volmer (SV) Plot Stern-Volmer (SV) plot is deduced to gain a first-hand sight regarding the nature of the quenching process. In the present context, under optimal conditions, linear SV plot is constructed (Fig. S2) (Lakowicz 1983). An important feature of SV plot is bimolecular quenching constant, which reflects the efficiency of quenching or accessibility of fluorophores to the quencher (Lakowicz 1983). Here, the following Stern-Volmer equation is used to deduce KSV, the Stern-Volmer quenching constant (F0/F) = 1+kqГ0[Q] = 1+KSV [Q] Where, F0 and F are the PL intensities in the absence and presence of quencher respectively. The Ksv value is found to be 5.42 × 10-6 M-1 and it is lower than the ideal value of diffusion-controlled quenching. This implies the occurrence of collisional quenching.
Fig. S2. Stern-Volmer (SV) plot for B. mori silk fibroin solution (BMSF) 6
6.2. Fluorescence microscopy Images collected under fluorescence microscope has also revealed similar results and exhibited PL quenching in presence of B12 (Fig. S3, A-D).
Fig. S3. Fluorescence micrographs of aqueous solution of B. mori silk fibroin showing high quenching in the presence of B12 under FITC and rhodamine monochromatic light (A and B); B. mori silk fibroin treated with B12 (BMSF + B12) (C and D).
7. Electrical measurements The dry silk cocoon membrane was reported as an insulator, but on absorbing moisture it can generate temperature dependent electrical current (Tulachan et al. 2014). Conducting polymer (PEDOT-PSS)silk fibroin bio composites was also used as electrochemical biosensors (Pal et al. 2016). Conductive polymers such as polypyrrole, polyaniline or poly 3,4-ethylene-dioxythiophene) and also combines with silk fibroin fibers to study the electrical properties (Xia and Lu 2008). The electrical properties are discussed in the plots as shown in Fig. S4, A-C.
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2
A
B
Impedance ()
Current (Amp)
4.00E-008
MF MF + milli Q water MF + B12 (dried)
3.00E-008
2.00E-008
1.00E-008
0.00E+000 0
1
2
3
4
0
5
1
0.00040
3
4
5
C
0.00035
Current (Amp)
2
Applied voltage (V)
Applied voltage (V)
MF MF + HBS
0.00030 0.00025 0.00020 0.00015 0.00010 0.00005 0.00000 0
1
2
3
4
5
Appied voltage (V)
Fig. S4. (A) Impedance versus applied voltage plot of MF, (B) IV plot for MF, MF + milli Q water, and (C) MF, MF+ HBS. 8. Dynamic Light Scattering (DLS) study
DLS studies are done for understanding the charge variation and size variation in absence and in presence of analyte B12 (Fig. S5). Zeta potential studies are done for BMSF, BMSF+B12 and B12 [Fig. S5 A]. The analysis of results show the variation in zeta potential with zeta potential values of BMSF and BMSF+B12 to be -3.55 mV and -2.75 mV respectively while that of B12 as -30.6 mV. Average size studies are further carried out. The average size of BMSF and BMSF+B12 are found to be 266.0 (d.nm) and 450.3 (d.nm) respectively while that of B12 as 300.4 (d.nm) [Fig. S5 B].
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-3.55
500000
Total Counts
400000 300000 200000
BMSF
100000
BM
0 -100
0
100
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100
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100
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Apparent Zeta Potential (mV)
-2.75
400000
Total Counts
300000
200000
BMSF + B12
100000
BM + B12
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-100
0 Apparent Zeta Potential (mV)
-30.6
700000 600000 T otal C ounts
500000 400000 300000 200000 100000
B B12 12
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A
-100
0 Apparent Zeta Potential (mV)
266
10 Intensity (Percent)
8 6 4
BMBMSF
2 0 0.1
1
10
100
1000
10000
1000
10000
Size (d.nm)
Intensity (Percent)
6
450.3
5 4 3
BMSF + B12
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Intensity (Percent)
15
300.4
10
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0 0.1
B12
B12
B
1
10
100
1000
10000
Size (d.nm)
Fig. S5. The variation of (A) Zeta potential and (B) Size 9
9. FT-IR spectral analysis The FTIR spectral analysis of the sample before and after interaction with B12 demonstrated no noticeable change in its peak positions (Fig. S6). The FT-IR spectra of the silk fibroin protein is previously described.
% T (a.u.)
MF+B12
MF
4000 3500 3000 2500 2000 1500 1000 -1
Wavenumber (cm )
Fig. S6. FT-IR spectra of MF and MF treated with B12 (MF+B12). 10. Selectivity and interference studies Selectivity study has been carried out with some biologically significant molecules like aspartic acid (Asp A), cysteine (Cys), glycine (Gly), L DOPA (L Dopa), phenylalanine (phe alanin), tryptophan (Tryp), retinol (Ret), bovine serum albumin (BSA), and cytochrome C (Cyt C). It is observed that the developed sensor did not exhibit appreciable sensitivity towards these biomolecules as shown in the Fig. S7A. In addition, a systematic study of interference of some amino acids aspartic acid (Asp A), cysteine (Cys), glycine (gly), tryptophan (Tryp), Phenyl alanine (Phe), Retinol (Ret), BSA, and biomolecules cytochrome C (Cyt C), L-DOPA, Hemin, and Hemoglobin were carried out as depicted in Fig. S7B. It is observed that these biologically significant molecules exhibited minimal interference to B12 sensing. A probable reason is attributed due to the absence of resonance energy transfer process between the sensor and analytes as depicted in Fig. S7C. In addition, in the case of electrical measurements, it has been found that the current-voltage characteristics of MF doesn’t show
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significant variation in the presence of similar concentration of riboflavin, retinol, hemin and hemoglobin as shown in the Fig. S7D.
A
B 60 50 40 30 20 10 0
0
Current (Amp)
D
Hemoglobin
Hemin
L- DOPA
Cytochrome C
BSA
Retinol
Phenylalanine
Tryptophan
nc .
Glycine
15
Cysteine
30
Aspertic acid
Co
% QE
45
%QE
Asp A Cys Gly L Dopa Phe alanin Tryp Ret BSA Cyt C B12
60
Different biochemicals
0.0000010 MF Riboflavin Retinol Hemin Hemoglobin
0.0000008 0.0000006 0.0000004 0.0000002 1.0
1.5
2.0
2.5
3.0
Applied voltage (V) Fig. S7. (A) Selectivity study of sensor BMSF toward various analytes, (B) Histogram depicting interference of BMSF based sensor with analytes, (C) Fluorescence resonance energy transfer (FRET) efficiency of sensor BMSF with different analytes. (D) Current-voltage plots of MF in the presence of riboflavin, retinol, hemin, and haemoglobin. 11. Calculation of limit of detection (LOD) Limit of Detection (LOD) is determined using the formula DL= 3.3 σ/S, where σ is the standard deviation of response and S is the slope of the calibration curve. It has been observed that PL quenching takes place upon incremental addition of analyte B12, so sensitivity can be characterized from the intercept of the plot of PL quenching against various concentrations of B12. The calibration curve has been constructed by plotting the PL quenching at different concentrations of B12 (Fig. S8).
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1.0 Y= (-0.0046)x + 1.07 R2= 0.97 SD= 0.23
I/Io
0.8
0.6
0.4
0.2
0
30
60
90
120
150
180
Concentration (nM) Fig. S8. Calibration curve for B12 detection with BMSF. Standard deviation (SD) is 0.23. 12. Calibration curve for electrical measurements Quantitative analysis of the electrical measurements was performed by plotting calibration curve for varying concentration of B12 in an aqueous medium as depicted in Fig. S9. The linear calibration curve with positive slop indicates that the current density in MF increases linearly with the concentration of B12. From this calibration curve, the LOD is calculated using the formula, LOD= 3.3 σ/S, where S and σ are the slope of the plot and standard deviation respectively. The LOD was calculated to be 17.8 ppm. 0.020
Current (mA)
MF + B12
0.015
Linear fit of MF +B12
0.010 0.005
y=(1.11)x + 0.0023 2 R = 0.98 SD= 0.006
0.000 0.000
0.005
0.010
0.015
0.020
-6
[B12] 10 g/l Fig. S9. Calibration curve for the detection of B12 with MF. Standard deviation (SD) is 0.006.
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