Supporting information

An ultrasensitive electrochemical sensing platform for Hg2+ based on

a density controllable metal-organic hybrid microarray

Lei Shia, Zhenyu Chua, Yu Liua, Wanqin Jina,* and Xiaojun Chenb

a State Key Laboratory of Materials-Oriented Chemical Engineering, College of

Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing

210009, P. R. China

b College of Sciences, Nanjing University of Technology, Nanjing 210009, P. R. China

This file includes:

1. Crystal structure of Ni(en)3Ag2I4 hybrid material2. The proposed formation mechanism of the hybrid microarray3. Four-probe method for determining the conductivity of the hybrid crystal4. The effect of different surface coverage of 1,4-benzenendithiol on the morphologies

of obtained hybrid microarray5. The electrochemical characterizations of the hybrid films6. EIS characterization of the fabricated Hg2+ biosensor7. Optimization of the experimental conditions8. The dosage of the nicking endonuclease used in the nicking reaction9. The consumption time in the nicking reaction10. Performances comparisons of Hg2+ biosensors based on hybrid films with different

morphologies11. Stability and reproducibility of the Hg2+ biosensorTable S1. Comparison of the assay performances of our Hg2+ biosensor with those

reported in the literatures

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1. Crystal structure of Ni(en)3Ag2I4 hybrid material

An asymmetric unit contains two crystallographically different Ag+ ions (labelled

Ag1 and Ag2), two crystallographically inequivalent I- anions (labelled I1 and I2), one

Ni2+ ion together with one diaminoethane ligand (Fig. S1A). The Ag1, Ag2 and I2

ions occupy the 2b Wyckoff positions, the Ni1 ion occupies the 2a Wyckoff position,

the I1 ion and the C1, C2, N1 and N2 atoms in the diaminoethane ligand occupy the

general positions. The Ag1 and Ag2 ions are correspondingly bound to one I2-type

and three I1-type iodide ions to form two types of AgI4 tetrahedral coordination

spheres with C3 point-group symmetry. Two types of AgI4 tetrahedra share a vertex,

each Ag1-type AgI4 tetrahedron is surrounded by four Ag2-type AgI4 tetrahedra and

vice versa. The three-dimensional (3-D) {Ag2I42-} framework, which consists of AgI4

tetrahedra, is shown in Fig. S1B and S1C, projected respectively along the

crystallographic c- and a-axes. The Ni2+ ion is coordinated to six N atoms, which

belong to three diaminoethane molecules, to form an octahedral coordination sphere

with C3 point-group symmetry. Such mononuclear species are filled in the cavity of

the 3-D {Ag2I42-} framework (Fig. S1D).

Fig. S1. (A) An asymmetric unit of Ni(en)3Ag2I4 (the hydrogen atoms in the

diaminoethane ligands are omitted for clarity, and the atoms marked with # are at the

following symmetry positions: #1 = 1-x+y, 1-x, z; #2 = 1-y, x-y, z; #3 = -1+x, -1+y, z;

#4 = -1+x, y, z; #5 = 1-x+y, 2-x, z; #6 = 2-y, 1+x-y, z). (B) and (C) The 3-D {Ag2I4}

framework in the crystal of Ni(en)3Ag2I4, projected along the crystallographic c- and

a-axes respectively. (D) Illustration of Ni(en)32+ ions filled in the cavity in the 3-D

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{Ag2I4} framework.

2. The proposed formation mechanism of the hybrid microarray

It is obviously that the formed SAM induced the oriented growth of hybrid

crystals on the Au substrate. Since the relatively aligned and erect SAM of 1,4-

benzenendithiol was prone to be assembled on the Au surface, and the –SH groups in

1,4-benzenendithiol molecules were likely to orient along [001], the binding of –SH

groups with Ag+ ions of the hybrid crystals will enable crystal growth only along the

[001] direction to give the highly oriented hybrid microarray, which is schematically

illustrated in Fig. S2. Meanwhile the higher coverage of the 1,4-benzenendithiol

would provide more binding sites, leading to a more crowded distribution of hybrid

microarray. In addition, there was no agent on the bare Au substrate to induce the

oriented growth of hybrid films, disordered hybrid microcrystals were obtained in this

condition and without the binding interactions from the SAMs, the obtained

disordered microcrystals were prone to peel from the Au surface.

Fig. S2. Schematic illustrations for the formation of highly oriented hybrid films on

the 1,4-benzenendithiol functionalized Au substrates. Hybrid crystals grow in the

[001] direction.

3. Four-probe method for determining the conductivity of the hybrid crystal

Four-probe method was performed to detect the conductivity of a single hybrid

crystal approximately, which could avoid disturbances of the contact resistance

existing in the two-probe method. The resistivity was calculated by the equation (1):

ρ=2 πS UI (1)

ρ=2×3 .14×0. 06×2. 986×103Ω ·cm=1. 125×103Ω ·cm

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σ=1ρ=8 .9×10−4 S⋅m-1

Where ρ is the resistivity, S is the average length between each probe, U is the applied

voltage, I is the obtained current and σ represents the conductivity.

Fig. S3. (A) Photograph (left) and schematic diagram (right) of the four-probe model.

(B) The calibration curve showing voltage versus current, the slope represented the

resistance (Ω).

4. The effect of different surface coverage of 1,4-benzenendithiol on the

morphologies of obtained hybrid microarray

Fig. S4. Hybrid microarray with different distribution densities on the Au substrates

immersed in the 1,4-benzenendithiol solution for (A) 2 h, (B) 6 h and (C) 10 h

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respectively.

5. The electrochemical characterizations of the hybrid films

Fig. S5. CV curves in 10 mM K3Fe(CN)6 containing 3 M KCl of (A) different hybrid

films: (a) hybrid microarray and (b) disordered hybrid films. (B) hybrid microarray

with different densities, immersion time with 1,4-benzenendithiol for (a) 2 h, (b) 10 h

and (c) 6 h.

6. EIS characterization of the fabricated Hg2+ biosensor

Electrochemical impedance spectroscopy (EIS) measurements were implemented

to monitor the impedance changes during the hybridization process. The impedance

spectra included a semicircle portion and a linear portion. The semicircle portion at

higher frequencies corresponded to the electron-transfer limited process, and the

linear portion at lower frequencies represented the diffusion-limited process. The

semicircle diameter equaled to the electron-transfer resistance, Ret. Because the stem-

loop structured cDNA possessed huge steric hindrance with negatively charged

backbone, the access of the redox probe of Fe(CN)63-/4- to the electrode surface was

inhibited. A very large semicircle domain was observed (curve a in Fig. S6A),

implying a very high electron-transfer resistance of the redox probe. After cDNA

hybridized with rDNA strand on the exposure of Hg2+, although the amount of

negatively charged DNA strands increased, a conformation of stand-up double strands

structure was formed, decreasing the steric hindrance. Accordingly, the access of the

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Fe(CN)63-/4- to the electrode surface became a bit easier, leading to a relatively small

semicircle domain, as shown in curve b in Fig. S6A. Upon the nicking reaction, the

further decrease of the semicircle domain was observed, resulting from the cleavage

of cDNA and dissociation of some rDNA strands (curve c in Fig. S6A). At this

moment, the steric hindrance and the electro-negativity of the DNA strands on

electrode surface towards the Fe(CN)63-/4- significantly decreased. In contrast, the

hybridization process in the absence of Hg2+ was also investigated (Fig. S6B). There

only slight signal changes were observed, indicating that the hybridization and

nicking reaction could not be activated without Hg2+.

Fig. S6. Nyquist plots in the Fe(CN)63-/4- solution of the Hg2+ biosensor at different

sensing procedures (A): in the presnece of 5 nM Hg2+, (B): in the absence of Hg2+. (a)

before, (b) after hybridizing with the rDNA and (c) after the nicking reaction.

7. Optimization of the experimental conditions

RuHex molecules, which can bind to the anionic phosphodiester backbone of

DNA via the electrostatic attraction, served here as electrochemical probes to estimate

the amount of DNA strands at the electrode surface. The cDNA immobilized on the

electrode surface could bring about the adsorption of large numbers of RuHex

molecules, leading to an enhanced readout signal. Chronocoulometry is a more

accurate electrochemical technique than CV to quantify the amount of cDNA. The

chronocoulometric signal, redox charge (Q) of RuHex, was proportional to the

amount of cDNA which was dependent on the immobilization time in cDNA solution.

In addition, the cDNA surface coverage density (Γss/molecules∙cm-2) could be

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calculated by the equation (2):

Γss=(Qss∙NA/nFA)(z/m) (2)

Here, n is the number of electron per molecule for reduction (n=1), F is the Faraday

constant (96485 C/mol), A is the real area of the working electrode (cm2, A was

estimated to be 0.08 cm2), m is the number of nucleotides in the DNA (m is 27 in

cDNA), z is the charge of the redox molecules (z=3) and NA is Avogadro’s number

(NA=6.02×1023/mol). Qss is the net capacitive charges of cDNA.

To estimate the real surface of the hybrid microarray, here, we assume a simplified

picture, in which the dimensions of hexagonal prisms are uniform, namely 25 μm in

height, 5 μm in side length (Fig. S7C), and the surface area A of oriented films can be

calculated by the equation (3). According to the equation, the area A is approximately

estimated to be 0.08 cm2, which is about ten times greater than that of its macro area,

and this large specific area is beneficial for applications in chemical sensing.

A=[6×length×height+ 3√32

×length2 ]×(number of hexagonal prisms )(3)

Fig. S7. (A) Distribution density of cDNAs on the surface of modified electrodes

incubated with 2 μM cDNA for 1 h, 3 h, 5 h, 7 h and 9 h respectively. (B) The

correlation between Δi and immobilization time in the cDNA solution (after the

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treatment with 10 nM Hg2+). (C) The cross-section of hybrid microarray on Au

surfaces after being immersed in the crystallization solution for 10 h.

8. The dosage of the nicking endonuclease used in the nicking reaction

As the poisonous Hg2+ may disturb the activity of NE, the dosage of the NE was

investigated in the work, as shown in Fig. S8. When the Hg2+ concentration was fixed

at 5 nM, the current signal of MB decreased quickly along with the dosage of NE

changed from 10 to 30 U during the initial nicking time. However, after the current

signal reached a relative plateau, the difference between the dosage of 20 U and 30 U

became less obvious. Similarly, the signal variation tendency was observed in the

presence of 500 nM Hg2+. Therefore, the preferential dosage of the NE was chosen as

20 U.

Fig. S8. Nicking time for the nicking reaction was recorded at different usage

amounts of nicking endonuclease of 10 U, 20 U and 30 U respectively. The Hg2+

concentrations in (A) and (B) were 5 nM and 500 nM.

9. The consumption time in the nicking reaction

The time for the nicking reaction was also optimized here, as shown in Fig. S9.

When 50 pM Hg2+ was added into the solution, the reaction rate was slow and the

corresponding current value reached a plateau after about 90 min. However, as for a

higher Hg2+ concentration of 500 nM, the reaction rate was accelerated and a steady

current was obtained after about 30 min. Thus, considering different reaction rates

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under different Hg2+ concentrations, the reaction time was optimized as 2 h for all the

detections.

Fig. S9. Nicking time for the nicking reaction was recorded at different Hg2+

concentrations of 50 pM, 500 pM, 5 nM, 50 nM, 100 nM, and 500nM respectively.

10. Performances comparisons of Hg2+ biosensors based on hybrid films with

different morphologies

Fig. S10. Comparisons in the performance of the Hg2+ biosensors based on the

ordered hybrid microarray and disordered hybrid film modified electrode.

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11. Stability and reproducibility of the Hg2+ biosensor

Fig. S11. (A) Stability and (B) reproducibility tests of the Hg2+ biosensor, 1 nM Hg2+

was added each time.

Table S1. Comparison of the electrochemical Hg2+ sensor performances

Sensors MethodDetection limit / nM

Linear range

Assay time[a] Reference

Iridium microelectrodes

ASV 0.5 5-50 nMca.15 min

[1]

Rotating disc gold electrode

ASV 0.05 0.2-400 nM ca. 5 min [2]

cDNA/gold electrode

DPV 0.5 1 nM-0.1

µMca. 2.5 h [3]

cDNA/graphene oxide

EIS 1 1-300 nM ca. 2 h [4]

cDNA/gold electrode

CV 0.6 0.001-10

µMca. 0.5 h [5]

cDNA/gold electrode

SWV 2.5 5.0 nM-1.0

µMca. 1h [6]

cDNA/gold electrode

ECL 0.25 0.5 nM-2.5

µMca. 5.3 h [7]

cDNA/gold nanoparticles-polydopamine-

carbon nanospheres

CV 0.0520.1 nM-1

µM- [8]

10

cDNA/Ni(en)3Ag2I4

microarraySWV 0.005

15 pM-0.5 µM

ca. 4 h This work

[a] Assay time is calculated from the injection of the Hg2+ to the readout of the

response signal.

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