Copper Oxide Assisted Cysteine Hierarchical Structures for Immunosensor Application Chandra Mouli Pandey, Gajjala Sumana, Ida Tiwari Supplementary Information EXPERIMENTAL SECTION Chemicals. Copper Nitrate (Cu(NO 3 ) 2 ·3H 2 O), Sodium hydroxide (NaOH), N,N-, Dimethyl acetamide (DMAC), L-Cysteine, N- hydroxy succinimide (NHS), N-ethyl-N-(3-dimethyl amino propyl carbodimide (EDC), Bovine serum albumin (BSA) and all other reagents and solvents are of analytical grade and have been procured from Sigma-Aldrich (India). Monoclonal antibodies and the target cells of E. coli (O157:H7) were procured from Kirkegaard & Perry Laboratories, Gaithersburg MD, USA. Phosphate buffered saline (PBS) was prepared by mixing PB solutions with 0.9% NaCl. Ultrapure water (Mill-Q, Millipore, 18.2 MΩ resistivity) was used throughout the experiments. Synthesis of copper oxide (CuO) nanoparticles. CuO nanoparticles were synthesised by the reported procedure with slight modifications. 1, 2 Briefly in a round-bottom flask equipped with a refluxing device, 18 mmol Cu(NO 3 ) 2 ·3H 2 O was dissolved in 120 ml of DMAC at 100 o C with vigorous stirring. Further, 60 ml NaOH (12mmol) solution in DMAC–water mixed solvent was rapidly added to the Cu(NO 3 ) 2 solution, until there is formation of a large amount of black precipitate. The solution was maintained at the crystallization temperature for 30 min and again heated at 120 o C (20 min). The formed black precipitate was then cooled to room
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Copper Oxide Assisted Cysteine Hierarchical Structures for Immunosensor ApplicationChandra Mouli Pandey, Gajjala Sumana, Ida Tiwari
spectroscopic measurements have been carried out using Perkin-Elmer spectrometer (Model
Spectrum BX) at 25oC. Electrochemical analysis has been conducted on an Autolab
potentiostat/galvanostat (Eco Chemie, Netherlands) using three-electrode cell with Au as
working electrode, platinum as auxiliary electrode and Ag/AgCl as reference electrode in
phosphate buffer (PBS, 100 mM, pH 7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6]3-/4-. The
impedance was performed with 10 mV sinusoidal modulation amplitude at an applied bias
potential of +0.23 V from 0.1–105 Hz frequency range at 12 steps per decade.
Figure S1. Schematic illustration for the preparation (a) CuO nanoparticle (b) CuCys and (c)
immunosensor fabrication.
Figure S2.1H NMR spectra of (i) CuCys and (ii) cysteine in 90% 2H2O/10%H2O solutions at
300 K under Ar atmosphere.
Figure S3. TEM image showing the effect of AuNP on the growth of Cys at (a) 10 mM
cysteine concentration and (b) 100 mM concentration. SEM image showing the
agglomeration of AuNPs on Cys at (c) 10 mM concentration and (d) 100 mM concentration.
Figure S4. EIS plot showing the change in Rct for (i) Cys/Au electrode and (ii) CuCys/Au
electrode and (iii) bare Au electrode.
Figure S5. Cyclic voltammogram analysis of (i) bare Au electrode (ii) CuCys/Au electrode
(iii) Ab/CuCys/Au electrode (iv) free site blocking of Ab/CuCys/Au electrode with BSA and
(v) E. coli cells/Ab/CuCys/Au electrode in phosphate buffer (PBS, 100 mM, pH 7.0, 0.9%
NaCl) containing 5 mM [Fe(CN)6]3-/4-.
Figure S6. Electrochemical impedance spectroscopy showing (a) influence of antibody
concentration and (b) influence of incubation time of E. coli cells on the variation of the
Δ1Rct.
Figure S7. Bar diagram showing the interference of non-E.coli bacterial pathogens on the
immunosensor.
Figure S8. (a) Studies on the stability of the immunosensor indicaing the % change in Rct of
the fabricated CuCys based imunosensor with storage time (in days). (b) Change in ΔRct of
the fabricated CuCys based imunosensor towards E. coli cells detection after each
regeneration cycle (6 cycles).
Table ST1. Infrared band assignments of L-cysteine and CuCys.
Table ST2. The fitting values of the equivalent circuit elements and the electron–transfer
kinetics for the fabricated CuCys/Au electrode and Ab/CuCys/Au electrode.
Table ST3. Comparison of analytical results obtained from the EIS system and plate count method.Table ST4. Comparison table showing the performance of the fabricated immunosensor with other electrochemical based immunosensor reported in literature for E. coli detection.
Figure S1.
Figure S2.
(i)
(ii)
Figure S3.
Figure S4.
0 50 100 150 200 250 300 350 400 450 5000
50
100
150
200
(i) cys/Au electrode (ii)CuCys/Au electrode (iii) Au electrode
(iii)(ii)
(i)
Zre/
-Zim
/
(b)(a)
(d)(c)1 μm
1 μm1 μm
1 μm
Figure S5.
Figure S5 shows the cyclic voltammograms for the [Fe(CN)6]3−/4− redox probe at a bare Au, CuCys/Au, Ab/CuCys/Au, blocking of Ab/CuCys/Au with BSA, and Eccell/Ab/CuCys/Au electrodes. After the modification of the bare gold electrode with self assembly of CuCys, there is a decrease in the peak current and an increase in the separation of the peak potentials (curve ii) compared to the voltammetric behaviour of the bare Au. On the immobilization of antibodies on CuCys/Au electrode, further decrease in the peak current (curve iii) was observed. Additional decrease in the peak current was observed on blocking the unoccupied sites with BSA (curve iv). Further, binding of Eccell to the immobilized antibodies (curve v) produced a remarkable decrease in the peak currents. These results are consistent with the changes observed in the electron-transfer resistance by EIS.
-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6-3.0x10-4
-2.0x10-4
-1.0x10-4
0.0
1.0x10-4
2.0x10-4
3.0x10-4
4.0x10-4
Potential (V)
Curr
ent (
A) (v)(iv)
(iii)
(ii)
(i)
Figure S6.
The immobilization of optimum concentration of antibody on CuCys/Au electrode using EDC-NHS chemistry was determined on the basis of the change in Δ1Rct [Δ1Rct=Rct(Ab)-Rct(CuCysNf/Au)] resulting from the binding of antibodies to CuCys/Au electrode (Fig. S6 a) it was observed that the Δ1Rct increased rapidly from 10µg/ml to 80µg/ml, and then reached a plateau at antibody concentrations higher than 80µg/ml and gets saturated at this concentration. Consequently, the optimum antibody concentration of 80µg/ml was used to construct the immunosensor. Further the incubation time of the Ab/CuCys/Au electrode with the E. coli cells were also considered. This was tested by varying the incubation time from 5 min to 30 min. Figure S6 (b) shows a rapid increase of relative Rct from 10 min to 25 min of incubation. However, only a slight increase in impedance with incubation time longer than 25 min has been noticed. This is attributed to the maximum binding of the E. coli cells and the antibody. Therefore, 25 min was used as optimized incubation time between E. coli cells and the antibodies in subsequent experiments.
Figure S7.
The selectivity of the immunosensors against three other bacteria (S. aureus and S.
choleraesuis) was evaluated by checking the impedimetric responses at the same concentration level that of E. coli (1×103, 1.0×106, and 1×109 cfu/ml) under the experimental conditions selected above (Fig. S7). Initially, we fabricated a series of electrodes (n=6) using E. coli antibodies and exposed them to suspensions of increasing concentration of Salmonella typhi, Shigella dysenteriae, and Vibrio cholera. When the bioelectrode was incubated with other bacterial cells, there was negligible change in Rct, indicating that there was no significant cross-reaction of the immunosensor with other bacterial species. These results demonstrated that the electron-transfer resistance as recorded reflected the interaction between the antibody and the target E. coli cells, therefore showing the specificity of the immunosensor for E. coli. The comparison of the biosensing parameters such as linear range and detection limit of the present work with the recent reports is shown in Table ST4.
3 6 90
20
40
60
80
100
120
140
160
180
200
Rct
Log concentration of bacterial cells (cfu ml-1)
Salmonelle typhi Shigella dysenteriae Vibrio cholera E. coli
Figure S8.
In addition, the storage stability of the immunosensor was tested by putting the Ab/CuCys/Au
electrode in a refrigerator (4 oC) and measuring its response towards E. coli O157:H7
detection every five days (Fig. S8 (a)). After one month, the impedimetric response of the
sensor remains 88 % of the initial value indicating that the immunosensor had acceptable
reliability and stability which may be due to the biocompatibility of CuCys, which maintain
the bioactivity of antibodies and other biomolecules. Further the regeneration step was
performed by immersion of the working bio-electrode for 10 min in glycine-HCl buffer (0.1
M, pH = 2.2) and washed with PBS solution to interrupt the antigen−antibody immune
complexes. Figure S8 (b) shows that there is an gradual decrease in ΔRct with the increase of
regeneration times which decreased obviously after regenerating the imunosensor for 6 times.
This may be due to the gradually shell off and denaturation of Eccells/antibody or the structure
of CuCys could be destroyed during continuous processed by a glycine-HCl buffer and
cleaning with the increase of regeneration times. The results demonstrated that the proposed
immunosensor could be regenerated and used for at least 6 times with relative standard