Copper Oxide Assisted Cysteine Hierarchical Structures for Immunosensor ApplicationChandra Mouli Pandey, Gajjala Sumana, Ida Tiwari

Supplementary Information

EXPERIMENTAL SECTION

Chemicals. Copper Nitrate (Cu(NO3)2·3H2O), 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(NO3)2·3H2O was dissolved in 120 ml of

DMAC at 100 oC with vigorous stirring. Further, 60 ml NaOH (12mmol) solution in

DMAC–water mixed solvent was rapidly added to the Cu(NO3)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 oC (20 min). The formed

black precipitate was then cooled to room temperature and the obtained products were

centrifuged at 15000 rpm for 5 min in anhydrous ethanol to remove the impurities, and

dried in air at room temperature (Fig. 1a).

Preparation of Cu(II) assisted three-dimensional cysteine flowers. For the preparation

of copper oxide modified three-dimensional cysteine nanoflowers (CuCys), firstly, 100 mM

aqueous solution of L- Cysteine (pH 8.0) was prepared using the reported method.3 Further

1.1 mg of the prepared CuO nanoparticles was added, and the solution was sonicated using

ultrasonic bath for about 5 minutes at 25 oC (Fig. 1b). The growth of CuCys was observed

by keeping the solution at 25 oC for 6 h. It was revealed that the optimum time for the

formation of CuCys is less than1 h.

Pre-treatment and fabrication of CuCys modified gold electrode. The gold (Au) electrode

(0.5 cm2 diameter) was washed in boiling 2.0 M KOH for about 1 h, following ultra-

sonication in Piranha solution (3:1 H2SO4/H2O2) for 10 min and the electrode was

subsequently washed in water for 10 min. The electrode was voltammetrically cycled and

characterized in 0.2 M H2SO4 from -0.5 V to -1.4 V (vs.Ag/AgCl) with scan rate of 0.10 V/s

until a stable cyclic voltammogram is obtained. To fabricate the CuCys monolayer films, the

Au electrode was dipped into the CuCys solution overnight (6 h) at 27oC after which, the

modified electrode (CuCys/Au electrode) was rinsed repeatedly with deionized water.

Preparation of antibody/antigen solution. E. coli (O157:H7) specific monoclonal antibody

solution was prepared in PBS buffer; pH 7.4 containing 0.1% sodium azide. The bacterial

cells were stored in 50% glycerol solution at -20 oC and the serial dilutions (101 to 1 x 109 cfu

ml-1) were prepared in PBS buffer prior to use.

Immunosensor Fabrication. The fabricated CuCys/Au electrode was activated by

immersion in a mixture of 2 mM EDC and 5 mM NHS for 1 h at 27 oC, in dark condition.

Subsequently, the modified electrode was incubated in 20 µl of a 0.1 mg/ml antibody solution

in a humid chamber overnight at room temperature. Finally, the electrodes were rinsed with

PBS solution to wash away the excess and unbound antibodies. The antibodies immobilized

electrodes were finally treated with 1% bovine serum albumin (BSA) to block the unspecific

binding sites followed by PBS washing (Fig. 1c).

Characterization. The structural and morphological investigations of CuCys have been

carried out using X-ray diffractometric (XRD, Cu Kα radiation, Rigaku, miniflax 2) and

transmission electron microscopic (TEM, Hitachi Model, H-800) studies. The scanning

electron microscopic (SEM) images have been recorded using a JEOLJSM-6700F field-

emitting scanning electron microscope (FESEM, 15 kV). Fourier transform infra-red (FT-IR)

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

deviation of 8.79%.

(a) (b)

Table ST1.

L-cys CuCyF Assignments3420 -OH stretch

3179 -NH stretch2982 3030 -CH stretch2889 -CH stretch2552 -SH stretch2079 2094 -CH stretch1587 1586 -NH3

+ deformation1429 1408 -CO stretch1346 1338 -OH deformation1297 1297 -OH stretch1196 1194 -CN stretch1140 -CO stretch1064 1030 -CO deformation942 -OH deformation867 847 -CH deformation822 -CH stretch753 -CH2 stretch692 675 -CS stretch638 -CS stretch, -COO- stretch538 540 -COO- stretch

Table ST2.

Sl.no

Name of the electrode

Solution resistance(Rs, Ω)

Charge transferresistance (Rct, Ω)

Capacitance(Cdl, µF)

Apparent electron transfer rate constant (10-4 cm s-1)

Exchange current per unit area (10-4A/cm2)

1 CuCyNf /Au 30.7 214.4 3.71 3.38 1.192 Ab/CuCyNf /Au 28.7 304.4 2.77 2.63 8.43

Immobilization matrices Detection Range Limit Of detection

References

Gold nanoparticle modified graphene paper

1.5x102–1.5x107 1.5x102 cfu/ml

4

Au nanoparticles coated SiO2 assembled on the fullerene , ferrocene and thiolated chitosan composite

3.2x101 to 3.2x106 cfu/ml 15 cfu/ml 5

self-assembled monolayers (SAMs)-modified gold screen-printed electrodes

5 -1.0×108 cfu/ml 3.3 cfu/ml 6

Inter-digitated array microelectrode 4.36x105 - 4.36x108 cfu/ml 106 cfu/ml 7

Indium tin oxide chip 6 x 104 - 6 x 107 cfu/ml 6 x 103

cfu/ml

8

Boron doped diamond 4 x 104- 6 x 106 cfu/ml 4x104

cfu/ml

9

Hyaluronan modified nanoporous

membranes

10-105 cfu/ml 1.0x101

cfu/ml

10

CuCys/Au electrode 1x10-1x109 cfu/ml 10 cfu/ml Present work

Table ST3.

Samples Immunosensor (cfu/ml)

Plate count method (cfu/ml)

Relative error (%)

1 2.0x102 2.1x102 4.762 8.4x102 8.1x102 3.703 6.0x103 6.2x103 3.224 6.6x104 6.4x104 3.125 3.4x106 3.5x106 2.87

Table ST4.

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