Selection and Characterization of DNA Aptamers for Electrochemical

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Selection and Characterization of DNA Aptamers for Electrochemical Biosensing of Carbendazim Mohammed M. Zourob, and Shimaa Hassan Hassan Eissa Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04914 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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Analytical Chemistry

1

Selection and Characterization of DNA Aptamers for Electrochemical

2

Biosensing of Carbendazim

3 4

Shimaa Eissaa, Mohammed Zouroba,b*

5

6

a

7

Road, Riyadh 11533, Saudi Arabia

8

b

9

12713, Saudi Arabia

Department of Chemistry, Alfaisal University, Al Zahrawi Street, Al Maather, Al Takhassusi

King Faisal Specialist Hospital and Research Center, Zahrawi Street, Al Maather, Riyadh

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11 12 13 14 15 16 17 18 19

*Corresponding author:

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[email protected]

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1 ACS Paragon Plus Environment


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Abstract:

3

This article reports a novel aptamer-based impedimetric detection of carbendazim, a commonly

4

used benzimidazole fungicide in agriculture. High affinity and specificity DNA aptamers against

5

carbendazim were successfully selected using systematic evolution of ligand by exponential

6

enrichment (SELEX). The dissociation constants (Kds) of the selected DNA aptamers after ten in

7

vitro selection cycles were characterized using fluorescence-based assays showing values in the

8

nanomolar range. The aptamer which showed the highest degree of affinity and conformation

9

change was used to fabricate an electrochemical aptasensor via self assembly of thiol-modified

10

aptamer on gold electrodes. The aptasensor exploits the specific recognition of carbendazim by

11

the aptamer immobilized on the gold surface which leads to conformational changes in the

12

aptamer structure. This conformational change alters the access of a ferrocyanide/ferricyanide

13

redox couple to the aptasensor surface. The aptasensor response is thus, measured by following

14

the increase in the electron transfer resistance of the redox couple using Faradic electrochemical

15

impedance spectroscopy. This method allowed a selective and sensitive label-free detection of

16

carbendazim within a range of 10 pg/ml-10 ng/ml with a limit of detection of 8.2 pg/ml . The

17

aptasensor did not show cross reactivity with other commonly used pesticides such as

18

fenamiphos, isoproturon, atrazine, linuron, thiamethoxam, trifluralin, carbaryl and methyl

19

parathion. Moreover, the aptasensor has been applied in different spiked food matrices showing

20

high recovery percentages. We believe that the proposed aptasensor is a promising alternative to

21

the currently used methods for carbendazim monitoring.

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1 2 3 4

Introduction

5

activity for protecting plants such as fruits, vegetables and cereals from pathogens. However, the

6

extensive use of pesticides in agriculture leads to their accumulation inside the plants which

7

represents a serious risk to human health. Because of the high stability of the benzimidazolic ring

8

in CBZ 1, it persists for long time in the soil and gets absorbed by the plants via the roots, seeds

9

or leaves entering the food chain. Studies

Carbendazim (CBZ) is the most widely used benzimidazole fungicide that shows long term

2-3

have shown the potential toxicity of CBZ on both

10

humans and animals. CBZ affects dehydrogenase and phosphatase activities 4, may cause

11

infertility in males 5, alters hormones concentrations and can cause disruption in the endocrine

12

system 6. Thus, the sensitive and accurate detection of CBZ in various food commodities has

13

become increasingly important for the protection of the public health.

14

Several traditional methods are currently used for the detection of the CBZ such as 7-9

, liquid chromatography coupled with mass spectrometry

10-11

15

chromatography

16

electrophoresis

17

spectroscopy

18

they cannot be used for on-site monitoring of CBZ because of the high cost and large size of the

19

used instruments as well as the long analysis time. Moreover, interference from other

20

components of the food complex matrix may not allow the precise detection of CBZ, thus sample

21

purification is usually required prior to analysis. Several electroanalytical methods have been

22

also used for the detection of CBZ exploiting its inherent electroactivity. The electrochemical

23

detection of CBZ has been reported using different electrode materials such as phosphorus-doped

24

helical carbon nanofibers

25

modified with graphene oxide/ carbon nanotubes hybrid material

12-13

19-20

, surface enhanced Raman scattering

and chemiluminescence

22

21

14-15

, fluorescence

16-18

, capillary

, UV-visible

. These methods are relatively sensitive; however,

, boron-doped diamond electrode


23 24

, glassy carbon electrodes or montmorillonite clay

25

,


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1

multi-walled carbon nanotubes-polymeric methyl red film modified electrode

2

cyclodextrin/graphene hybrid nanosheets

3

powerful than other assays due to their low cost, ease of use, sensitivity and short analysis time.

4

However, other electrochemically active pesticides which are used concomitantly with CBZ in

5

various fields such as isoproturon

6

analysis. Therefore, the selective detection of CBZ in real samples is hardly achievable using the

7

reported electrochemical sensors that does not use a specific recognition receptor. Itak et al.

8

have reported the production of specific antibodies against CBZ and its application in magnetic

9

particle-based immunoassay. Enzyme linked immunosorbent assay (ELISA) kit for CBZ is

10

commercially available. The immunoassays have better selectivity and capability of high

11

throughput analysis of CBZ. However, the production of the antibodies for CBZ and other

12

pesticides is challenging as they are small molecules which have to be conjugated with carrier

13

proteins for immunization 31. Moreover, the haptens which are usually used as immunogens and

14

for coating in ELISA have to be synthesized from CBZ analogs in order to attach appropriate

15

function groups to the molecule to enable the conjugation with the protein

16

high cost and low stability of the antibodies limits the wide applicability of the immunoassays

17

for CBZ detection. Therefore, more stable, specific and cost effective recognition receptor for

18

CBZ is strongly needed to be employed in biosensing platforms in order to replace the old

19

technologies.

28

27

and

. The electrochemical methods appeared to be more

and fenamiphos

29

can interfere with CBZ complicating the

32

30

. Additionally, the

20

Aptamers are synthetic single stranded DNA (ssDNA) or RNA which can be identified

21

using in vitro selection protocol. Aptamers have appeared as promising alternatives to the

22

traditional antibodies showing high affinity and specificity. Unlike antibodies, aptamers are

23

highly stable at any condition and can be easily synthesized with very low cost. They can be also


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selected against low molecular weight analytes which are hard to produce antibodies for. Despite

2

the successful selection of aptamers against various small molecules

3

hormones37-38 and antibiotics

4

reported on the use of aptamers for the detection of organophosphorous pesticides 41-43. Here, we

5

report for the first time the selection of high affinity and specificity DNA aptamers for CBZ. The

6

application of the selected aptamer in a label-free biosensor using electrochemical impedance

7

spectroscopy detection is also presented. The combination between the high specificity and

8

affinity of the new aptamer and the sensitivity of the impedance technique enabled the rapid, low

9

cost and reliable detection of CBZ.

39-40

33

such as toxins

34-36

,

, aptamers for pesticides are very rare. Few studies have been

10 11

EXPERIMENTAL SECTION

12

Materials and reagents

13

Carbendazim, potassium ferricyanide (K3Fe(CN)6), potassium ferrocyanide (K4Fe(CN)6),

14

methanol, sodium chloride, potassium dihydrogen orthophosphate, 6-Mercapto-1-hexanol

15

(MCH), dipotassium hydrogen orthophosphate, acetonitrile and magnesium chloride were

16

obtained from Sigma (Ontario, Canada). Atrazine, linuron, thiamethoxam, trifluralin, carbaryl,

17

methyl parathion, fenamiphos and ioproturon were obtained from the food safety laboratory

18

(Qassim, KSA). The polymerase chain reaction (PCR) primers and the DNA library were

19

custom-synthesized by Integrated DNA Technologies Inc. (Coralville, USA). The thiol modified

20

aptamers

21

/SH/CGACACAGCGGAGGCCACCCGCCCACCAGCCCCTGCAGCTCCTGTACCTGTGTG

22

TGTG/-3'

23

/SH/GGGCACACAACAACCGATGGTCCAGCCACCCGAATGACCAGCCCACCCGCCAC

(CZ12:

and

5´-C6

CZ13:


5´-C6


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1

CCCGCG/ -3') were synthesized by Metabion (Planegg, Germany). CBZ ELISA test kit was

2

obtained from Bioo Scientific (Austin, USA). Taq plus DNA polymerase, urea, Tris-base, boric

3

acid, acrylamide/bis-acrylamide (40 % solution) and EDTA disodium dehydrate were obtained

4

from Bioshop Inc. (Ontario, Canada). Cellulose acetate centrifuge tube filters (pore size: 0.45

5

µm) were purchased from Corning life sciences (Tewksbury MA, USA). TOPO TA Cloning Kit

6

with One Shot MAX Efficiency DH5α-T1 was obtained from Invitrogen (NY, USA). Amicon

7

Ultra-0.5 mL centrifugal desalting filters with a 3kDa molecular cut-off were purchased from

8

EMD Millipore (Alberta, Canada). The CBZ solution was prepared by dissolving in methanol

9

(3µg/ml) and then diluted with binding buffer in order to obtain the working solutions. All the

10

pesticide solutions which were used for cross reactivity were prepared in acetonitrile (1mg/ml)

11

and diluted with binding buffer. Tris-EDTA buffer (TE buffer) is 10 mM Tris, pH 7.4, 1 mM

12

EDTA. The binding buffer which was used for the SELEX and detection experiments consists of

13

50 mM Tris, pH 7.5, 150 mM NaCl and 2 mM MgCl2. The Elution buffer consists of 7 M urea in

14

binding buffer. A phosphate buffered saline PBS solution (10 mM, pH 7.4) was used to dissolve

15

the [Fe(CN)6]3−/4− redox couple for the electrochemical measurements. The mango juice, soya

16

milk, tomato and plum samples were obtained from local store (Riyadh, KSA). All the solutions

17

were prepared using Milli-Q grade water.

18

Instrumentation

19

The ultraviolet-visible (UV) and fluorescence measurements were recorded using

20

NanoDrop 2000C Spectrophotometer and NanoDrop 3300 Fluorospectrometer, respectively

21

(Fisher Scientific, Canada). The ELISA measurements were performed using microplate reader

22

(Sunnyvale, USA). Electrochemical experiments were carried out using an Autolab

23

PGSTAT302N potentiostat (Eco Chemie, The Netherlands) controlled by 1.11 NOVA software.


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1

A three-electrode cell was used for all the electrochemical measurements, consisting of a

2

standard gold working electrode, an Ag/AgCl reference electrode and a platinum wire as counter

3

electrode.

4 5

Methods

6

In vitro selection protocol of the DNA Aptamer for CBZ

7

The selection of the aptamer was carried out using the protocol reported previously

34

. A

8

random ssDNA library (1.8 × 1015 oligonucleotides) was used for the aptamer selection which

9

consists of a central random region of 60 nucleotides flanked by two primer annealing sites of 18

10

nucleotides

at

the

3’

and

5’

ends

(5’-ATACCAGCTTATTCAATT

11

AGATAGTAAGTGCAATCT-3’). The DNA library was dissolved in binding buffer. A

12

pretreatment of the DNA solution was done before each round by heating it to 90°C for 5

13

minutes, then cooling at 4°C for 10 minutes and keeping at 25°C for 5 minutes. Two hundred µl

14

of the pretreated DNA which contain 3 nmol DNA in the first round and 150 pmol in the

15

subsequent rounds were added to the CBZ-bovine serum albumin (CBZ-BSA) coated wells of

16

the ELISA microtiter plate. Two wells were used for each round and 100 µl DNA solutions were

17

added to each well and incubated with mild shaking for 2 h. at room temperature (the incubation

18

time was gradually decreased in the subsequent rounds in order to increase the stringency of the

19

selection as shown in Table S1). After that, the unbound and the weekly bound DNA to CBZ

20

were removed by washing the wells for 3 times with binding buffer. Then, the bound DNA to

21

CBZ was eluted in the first seven rounds by incubation with preheated 100µL elution buffer in

22

each well for 6 times with continuous heating at 90°C for 10 minutes. After the seventh round,

23

specific elution was performed (the bound DNA was eluted by incubation with 10 mM CBZ


-

N60-


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1

solution for 30 min.) and the binding time was decreased from 45 to 30 min. Then, the eluted

2

aliquots were concentrated and desalted by ultrafiltration using Amicon filters. The purified

3

DNA pool after elution was then PCR amplified using 15 parallel reactions of 75µL each

4

contains 2 units of Taq Plus polymerase enzyme, Taq buffer, 2mM MgCl2, 200µM dNTP,

5

0.2µM of forward and reverse primers. The primers sequences were modified with fluorescein

6

and a polyethylene glycol (PEG) spacer linked to poly-A tail as reported previously

7

Forward primer is 5’-fluorescein-ATACCAGCTTATTCAATT-3’ and reverse primer is 5’-

8

poly-dA20-PEG6-AGATTGCACTTACTATCT-3’. PCR thermal cycle conditions were 94°C for

9

10 minutes, followed by 25 cycles of 94°C for 1 minute, 47°C for 1 minute, 72°C for 1 minute,

10

and a final extension step of 10 minutes at 72°C. The double stranded DNA (dsDNA) solution of

11

the product PCR was dried by SpeedVac, then resuspended in 50:50 water and formamide and

12

heated to 80°C for 2 minutes. Denaturing polyacrylamide gel electrophoresis (PAGE), 12% was

13

used to separate the relevant fluorescent ssDNA strand (aptamer) from the dsDNA PCR product.

14

The fluorescent DNA was then eluted from the gel band by freeze-thaw cycle in TE buffer (the

15

gel slices were crushed into small pieces using a syringe and suspended into 3 ml of TE buffer,

16

then frozen at -80 oC for 10 mins, thawed at 60 oC for 5 min and then heated at 90 oC for 5 min.).

17

The product was then concentrated, desalted, quantified by measuring the UV using Nanodrop

18

device and used for the subsequent selection cycle.

34,36

.

19

Cloning and sequencing of the selected DNA for CBZ

20

After ten selection rounds when the recovered DNA began to plateau, the SELEX was stopped

21

and the obtained DNA pool from the last round was PCR amplified with unmodified primer set

22

and cloned into pCR2.1-TOPO vector using TOPO TA Cloning Kit. The E Coli competent cells

23

were grown on LB-agar plates supplemented with ampicilin, X-Gal and IPTG. Positive colonies


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1

(white color) were picked and grown in LB liquid media. The ssDNA inserts (aptamers) were

2

amplified using the M13 forward and reverse primer sites within the vector and sequenced. The

3

identified CBZ specific aptamers were the aligned via PRALINE 44 software Fig. S1.

4 5

Binding assays of the CBZ aptamers using fluorescence detection

6

The fluorescence binding affinity studies were realized following the previously reported

7

protocol 34. Six of the sequenced aptamers were synthesized with fluorescein modification at the

8

5' end after eliminating the primers sequences and their binding affinities to CBZ were tested.

9

First, the aptamers were pretreated by heating and cooling as previously described in the SELEX

10

part. Then 100 µl solution of each aptamer of different concentrations (25, 75, 100, 150, 300 nM)

11

in binding buffer were incubated on the CBZ-BSA coated wells and incubated for 30 min. with

12

mild shaking at room temperature and protected from light. After binding, the wells were washed

13

with binding buffer and then the bound DNA aptamers to the CBZ were eluted using preheated

14

elution buffer at 90 oC for 10 min. The DNA library was used as a control. The assay was

15

performed in duplicate.

16

In order to determine the dissociation constants of the aptamers with CBZ, binding assays were

17

performed as described above, but using different concentrations of the fluorescent aptamers (25

18

to 300 nM). Then, the fluorescence intensity of the eluted DNA in each sample was measured

19

and used to plot the binding curves. The Kd for each CBZ aptamer was determined by non-linear

20

regression analysis of the binding curve.

21

CBZ aptasensor fabrication

22

Before modification, standard gold electrodes (2 mm in diameter) were polished with alumina

23

slurries with various particle sizes (1.0, 0.3, and 0.05 µm), and washed with Milli-Q water. Then



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1

the electrodes were cleaned with piranha solution, followed by cyclic voltammetry (CV)

2

cleaning in 0.1 M H2SO4 by scanning between 0.2 and 1.6 V for 5 min, then washed with

3

ultrapure water and dried under nitrogen stream. The cleaned electrodes were then incubated in 1

4

µM of thiol modified CZ13 or CZ12 aptamers in binding buffer for 24 h. After the

5

immobilization, the electrodes were washed with Tris-HCl, pH 7.4 and then incubated with 1

6

mM MCH in PBS buffer for 30 min. The aptamer-modified Au electrodes were rinsed

7

thoroughly with binding buffer and used immediately in the electrochemical experiments or

8

stored in binding buffer solution at 4 °C.

9

Electrochemical measurements

10

For the CBZ detection experiments, 10 µL droplet of CBZ solution of certain concentration

11

(0.01, 0.1, 1.0, 10, 100, 100 and 1000 ng/ml) in binding buffer were incubated onto the

12

aptasensor surface for 30 min. After incubation, the electrodes were washed with Tris-HCl, pH

13

7.4 buffer to remove the nonspecifically bound molecules and then the electrochemical

14

measurements were recorded. The selectivity experiments were performed by incubating the

15

aptasensors with 100 ng/ml of atrazine, linuron, thiamethoxam, trifluralin, carbaryl, methyl

16

parathion, fenamiphos, ioproturon or CBZ. The sensors were then washed and subjected to the

17

measurements.

18

The CV experiments were performed at a scan rate of 100 mV/s. The electrochemical

19

impedance spectroscopy (EIS) measurements were recorded over a frequency range from 10 kHz

20

to 0.1 Hz using an alternative voltage with amplitude of 10 mV, superimposed on a DC potential

21

of 0.20 V (vs Ag/AgCl reference electrode). The impedance data were plotted as Nyquist

22

diagrams with a sampling rate of 25 points per decade. The impedance spectra were fitted using


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1

NOVA 1.11 software. All the electrochemical measurements were recorded in 5 mM

2

[Fe(CN)6]3−/4− redox couple solution (1:1 molar ratio) in a 0.1 M PBS buffer.

3 4

Food samples analysis

5

Preparation of extracts

6

Four commercial samples obtained from local stores were used (mango juice, soya milk, tomato

7

and plum). For the solid fruit samples (tomato and plum), the samples were blended to obtain a

8

homogeneous mixture. One gram of each sample was mixed with 2 ml of CBZ extraction buffer

9

from the ELISA kit, 4 ml of acetonitrile and vortexed for 3 min. Then, the mixture was

10

centrifuged for 10 min at 4000 g. 1 ml of the supernatant was transferred to another tube and

11

dried by evaporating the solvent in 60 oC water bath. The dried residue is then dissolved in 1 ml

12

solution of either binding buffer for the aptasensor experiments or extraction buffer for the

13

ELISA experiments. For the liquid samples (juice and milk), the samples were centrifuged at

14

4000 g for 10 min., and the supernatant was filtered using 0.45 µm cellulose acetate centrifuge

15

filters. The prepared extracts were divided into small aliquots and stored at -20 °C until used.

16

Finally, the extracts were diluted 1:10 with extraction buffer for ELISA or binding buffer for the

17

aptasensor and spiked with two concentrations of CBZ to obtain 1 ng/ml and 10 ng/ml. The

18

spiked extracts were then tested on the aptasensors as described above for the standard solutions.

19

Competitive ELISA experiment for comparison study

20

The extracts were analyzed according to the protocol of the ELISA kit. Briefly, 50 µL of the

21

spiked extracts and the five standard solutions (negative, 0.5, 1.5, 6, 12 ng/mL) provided in the

22

ELISA kit were added into the CBZ-BSA coated wells. Then, 100 µl of the first antibody for

23

CBZ was added to the wells and incubated for 30 min. Then, the wells were washed and



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1

incubated with 150 µl of second antibody-HRP conjugate for 30 min followed by washing the

2

wells and adding the TMB substrate. After 15 min, the reaction was stopped using stop buffer

3

solution and the absorbance of the ELISA plate was measured in plate reader at 450 nm wave

4

length. All the samples were done in duplicates.

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5 6

Results and discussion

7

Selection of DNA aptamers for CBZ

8

One of the most important steps for the aptamer selection is the immobilization of the small

9

target analyte on a solid support in order to enable the separation of the bound from the unbound

10

DNA during the selection. Because of the very small size of CBZ (191 g/mol), the lack of

11

chemical function groups and the need of sophisticated organic synthesis process to produce a

12

hapten for conjugation 32, we utilised a commercial ELISA plate on which CBZ-BSA conjugate

13

was already coated as a solid matrix. The progress of the SELEX was followed by assessing the

14

DNA recovery after each round by measuring the fluorescence intensity of the eluted DNA. As

15

shown in Fig. 1A, no fluorescence was detected from the first until the fifth round which

16

indicates that the concentration of the recovered DNA was very low. At round 6, the DNA

17

recovery started to increase. Since the BSA protein size is much larger than the CBZ molecule,

18

there is a high possibility that the DNA could bind to the BSA leading to selecting aptamers that

19

are not specific to CBZ. Therefore, in order to discriminate between the bound DNA to CBZ and

20

to the BSA, we used a specific elution step from round 8 to 10 to ensure the recovery of only the

21

CBZ specific sequences. In fact, using specific elution by affinity is recommended in all the

22

selection cycles. However, this method requires high concentration of ultra pure analyte which

23

increases the cost. Therefore, simple non specific elution using denaturation with urea and


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1

heating is usually performed in the first rounds until high recovery of DNA is observed. Then,

2

specific elution is used in the last few rounds to specifically recover the sequences which have

3

the capability to bind to the free CBZ in solution. After the specific elution, extensive clean-up to

4

remove the CBZ from the DNA was performed using ultrafilteration as the excess CBZ can

5

inhibit the PCR.

6

After cloning of the eluted DNA from round 10, we picked 20 clones for sequencing and 44

7

all sequences were identified. Using PRALINE

software, multiple sequence alignment was

8

performed in order to see the similarities in the obtained sequences. As shown in Fig. S1, highly

9

conserved consensus sequence motifs were observed, therefore, we divided the sequences into 6

10

groups. We chose a representative sequence from each group for the affinity testing to CBZ.

11

Fig. 1B shows that the six tested sequences (CZ1,CZ2,CZ12,CZ5,CZ6 and CZ13) exhibited high

12

affinity to CBZ compared with the initial DNA library (control). Fig. 2 shows the binding

13

affinity curves of the six tested sequences. Non linear regression fitting of the binding curves was

14

used to determine the Kds. As shown in Table 1, all the tested sequences exhibited kds in the low

15

nanomolar range (60-250 nM) which indicates high affinity of the selected aptamers for CBZ.

16

The aptamer sequences which showed the highest affinity to CBZ was CZ12 and CZ13.

17 18

Electrochemical aptasensor for CBZ

19

The two aptamers which showed the highest affinities to CBZ (CZ12 and CZ13) were

20

synthesized with thiol modification from the 5' terminals and then immobilized on the gold

21

electrodes via self assembly monolayer formation. Our goal was to test the possibility of utilizing

22

the selected aptamers on a label-free biosensor platform based on probing the change in the

23

electrochemical impedance spectra of [Fe(CN)6]4-/3- redox couple of the aptasensor upon binding.



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1

First, to fabricate the aptasensor, the aptamers were immobilized on the gold electrode

2

surface via self assembly and the free gold surface was blocked with MCH. As shown in Fig. 3,

3

CV and EIS were used to characterize the stepwise modification of the aptasensor. Fig. 3A

4

shows the CV of the bare Au, aptamer-modified Au and after blocking with MCH measured in a

5

5 mM solution of [Fe(CN)6]4-/3- redox couple in PBS buffer, pH 7.4. As expected, the clean bare

6

gold electrode showed a reversible redox peaks with a peak to peak separation (∆E) of 80 mV.

7

However, after the aptamer self assembly, the ∆E increased and the anodic and cathodic peak

8

currents decreased. This change of the characteristics of the CV indicated the successful

9

attachment of the aptamer on the gold which lead to shielding of the gold surface with the

10

negatively charged DNA. This shielding effect has lead in turn to diminishing of the electron

11

transfer between the redox molecules and the electrode. Upon blocking the Au electrodes with

12

MCH, a further increase in the ∆E was seen which suggests a decrease in the electron transfer

13

rate of the redox couple likely due to covering some of the free pinholes on the surface with the

14

MCH. In fact, the blocking step with MCH in order to form a mixed monolayer with the

15

thiolated aptamers is a crucial step not only to minimize the non specific adsorption but also to

16

avoid the physical adsorption of the DNA on the surface and retain the conformation of the

17

aptamers on the surface

18

work at 1µM concentration.

34

Page 14 of 33

. Therefore, the MCH concentration was optimized in our previous

19

The fabrication of the aptasensor was also confirmed using EIS as shown in Fig. 3B. The

20

impedance spectra were represented as Nyquist plots which exhibit a semicircle in the high

21

frequency domain and a straight line in the low frequency domain. The semicircular part

22

represents the electron transfer and the capacitive behavior of the system and the linear part

23

corresponds to the diffusion process. A modified Randles circuit was used to fit all the


Page 15 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

impedance spectra (inset of Fig. 3B, fitting is shown in Fig. S2). This circuit consists of four

2

elements, charge-transfer resistance (Rct), constant phase element (CPE), solution resistance (Rs),

3

and Warburg impedance (Zw).

4

with a distinctive straight light which indicates the fast electron transfer kinetics of the clean gold

5

surface. However, after the incubation of the thiolated aptamer on the electrodes, a significanmt

6

increase in the diameter of the semi circle of the impedance spectra was observed. This increase

7

in diameter indicates the increase in the Rct due to the reulsion of the [Fe(CN)6]4-/3- anions with

8

the attached negatively charged DNA. Then the Rct was further increased upon incubation with

9

MCH. Thus, the EIS results were consistent with the CV results indicating the successful

10

45-46

The EIS of the bare Au electrode showed a small semi circle

fabrication of the aptasensors.

11

After characterizing the aptasensors, we then started to test the response of the aptasensor

12

to the binding with CBZ. Two aptasensors were individually prepared by immobilizing the

13

aptamers; CZ12 and CZ13 on two different electrodes and the response of the two sensors to the

14

binding with CBZ was assessed. As shown in Fig.S3, the response of the aptasensors, measured

15

as the change of the Rct upon binding with 100 ng/ml solution of CBZ, was significantly higher

16

in the case of CZ13. This result demonstrates that the aptamer CZ13 exhibits more change in the

17

conformation upon binding with CBZ than CZ12 despite of their comparable Kds. The change of

18

the aptamer conformation has lead to more shielding of the Au surface, less access for the

19

[Fe(CN)6]3−/4− redox molecules to surface and therefore, retardation of the electron transfer. The

20

change of the Rct due to the conformation change induced by the CBZ binding to aptasensor

21

CZ13 was used as the basis for the biosensor detection (scheme 1).

22

In order to determine the analytical range of the aptasensor, the impedance response of

23

the aptasensor after binding with different concentrations in the range of (10 pg/ml to 1 µg/ml) of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 33

1

CBZ was monitored. As shown in Fig.4A the diameter of the semi circle of the impedance

2

spectra increases with increasing the CBZ concentration. The Rct values obtained from the

3

impedance spectra was used to build the calibration curve as it was the most changeable

4

parameter in the circuit. The aptasensor response was calculated as the percentage change in the

5

Rct after binding with certain concentration of CBZ ((Ro-R)/Ro%). Fig. 4B shows the calibration

6

curve of the aptasensor (a plot of the aptasensor response versus the logarithm of the CBZ

7

concentration). A linear relationship was observed from 10 pg/ml to 10 ng/ml (the linear

8

regression equation is: (Ro-R)/Ro %= 62.2+ 22.9 log C [ng/ml], R = 0.994. All the measurements

9

were done in triplicates and the error bars represent the standard deviations of the measurements

10

(RSD%= 3.0-5.4), demonstrating the high accuracy of the aptasensor. The limit of detection

11

(LOD) of the developed CBZ aptasensor was 8.2 pg/ml (LOD= 3Sb/m, where Sb is the standard

12

deviation of the blank signal and m is the slope of the linear part of the calibration curve). This

13

calculated detection limit indicates high sensitivity of the proposed aptasensor as it is lower than

14

the LOD of the available commercial ELISA kit by 2 order of magnitude, the reported

15

immunoassay 30, Raman scattering detection 14 as well as electrochemical assays 22,27,47-48 (Table

16

S2).

17

The cross reactivity of the developed CBZ aptasensor with 100 ng/ml solution of a

18

variety of potential related interfering agricultural compounds was tested in order to verify the

19

selectivity of the sensor. As show in Fig. 5, the aptasensor responses with all the examined non

20

specific pesticides (atrazine, linuron, thiamethoxam, trifluralin, carbaryl, methyl parathion,

21

fenamiphos and ioproturon) were not significant compared with the high response obtained with

22

CBZ. These results confirm the high selectivity of the developed aptasensor and its possible


Page 17 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

applicability for the selective quantification of CBZ without interference with other compounds

2

particularly other electroactive molecules such as fenamiphos and ioproturon

3

The developed aptasensor was then examined in different real food samples (soya milk,

4

mango juice, tomato and plum fruit). After extraction as explained in the experimental section

5

the samples were spiked with two different concentrations of CBZ (1.0 and 10 ng/ml). The

6

spiked extracts were then analysed using the proposed aptasensor and a commercial ELISA kit.

7

As shown in Table 2, the recovery percentages of the proposed aptasensor were between 89%

8

and 95% which were in good correlation with that obtained using standard ELISA. These results

9

reveal that there is no significant matrix effect on the aptasensor response signal, and thus, the

10

aptasensor can be applied for the detection of CBZ in complex real food samples for routine use.

11 12 13 14 15

Conclusion In this work, we presented the first selection and characterization of DNA aptamer sequences

16

against CBZ. The selected aptamers exhibited high affinity for CBZ with calculated dissociation

17

constants in the nanomolar range. One of the highest affinity aptamers (Kd=65 nM) was then

18

applied in a label-free biosensor using EIS. The detection mechanism was based on monitoring

19

the change in the charge transfer resistance of a [Fe(CN)6]3−/4− redox couple that is induced by

20

the alteration of the aptamer conformation upon binding with CBZ. The aptasensor showed fast

21

response (30 min) and high sensitivity (detection limit of 8.2 pg/ml). Moreover, the developed

22

aptasensor exhibited highly selectivity to CBZ and did not show any cross reactivity with other

23

pesticides such as atrazine, linuron, thiamethoxam, trifluralin, carbaryl and methyl parathion,

24

particularly electroactive compounds (fenamiphos and ioproturon). The aptasensor was also

25

applied for the detection of CBZ in spiked real food sample and high recovery percentages were



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

obtained which were in good agreement with the results obtained from a standard ELISA

2

method. This indicates great promise for using the developed aptasensor as alternative to the

3

current traditional assays.

4 5 6

ACKNOWLEDGMENTS

7

The authors would like to thank the food safety laboratory in Al-Qassim, KSA for providing the

8

standard pesticide solutions.

9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

References


Page 18 of 33

Page 19 of 33

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1498.

Page 22 of 33

Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications,

8 9

10 11 12 13

14 15 16 17 18 19 20 21

Figure captions:

22

Fig. 1 (A) The recovery profile of the eluted DNA during the SELEX cycles (a plot of the

23

fluorescence intensity versus the round number) (B) Fluorescence binding assays of the selected


Page 23 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

aptamers and the DNA library as a control. The error bars represent the standard deviations of

2

the measurements.

3

Fig. 2 Binding affinity curves obtained using fluorescence assays of CBZ with the aptamers and

4

CZ13 (A), CZ2 (B), CZ5 (C), CZ6 (D), CZ1 (E) and CZ12 (F). The Dissociation constants were

5

calculated by non linear regressing fitting of the curves. The error bars represent the standard

6

deviations of the measurements.

7

Fig. 3 CV (A) and EIS Nyquist plots (B) of 5 mM [Fe(CN)6]4−/3− redox solution in PBS buffer,

8

pH 7.4, for bare the Au electrode (black line), aptamer/Au (red line) and MCH/aptamer/Au (blue

9

line). The CV was done at scan rate of 100 mV/s and the EIS was recorded using a frequency

10

range of 104 to 0.1 Hz, a DC potential of +0.20 V and AC amplitude of 5 mV. The inset in Fig.

11

3B is the modified Randles equivalent circuit used to fit the impedance spectra.

12

Fig. 4 (A) Nyquist plots of the CBZ aptasensor after incubation with different concentrations of

13

CBZ 0.00, 0.01, 0.1, 1.0, 10 and 100 ng/ml. (B) The calibration curve for CBZ (plot of (Ro-

14

R)/Ro% vs. logarithm of the CBZ concentration. The measurements were done in triplicates and

15

the error bars represent the standard deviations of the measurements.

16

Fig.5 Comparison of the aptasensor response to 100 ng/ml solution of CBZ, atrazine, linuron,

17

thiamethoxam, trifluralin, carbaryl, methyl parathion, fenamiphos and ioproturon.

18

19 20

Scheme 1. The detection mechanism of the CBZ aptasensor based target-induced conformation

21

change of the aptamer.

22



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Table 1. Sequences and dissociation constants (Kd) of CBZ and selected aptamers.

3

Table 2. Real food samples testing using the developed CBZ aptasensor and commercial ELISA.

Page 24 of 33

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Table. 1

18

Aptamer

Aptamer sequence 24 ACS Paragon Plus Environment

Kd

Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


number

(nM)

CZ1

GGTCGCAGCAGCCTGTACACCCACCCACCAGCCCCAGCATCATACCCATTC GTGCCGTG

91

CZ2

GGCGACACAGCAACACACCCACCCACCTGCCCCATCAACAACCTATCCCTG TCCGTGCG

72

CZ12 CZ5

CGACACAGCGGAGGCCACCCGCCCACCAGCCCCTGCAGCTCCTGTACCTGT GTGTGTG GGCCATCGGACCACAGTACCCACCCACCGGCCCCAACATCATGCCCTCGTT GTTGTGTG

60.2 250

CZ6

CGACACGCAGCATACGTACGCCGCCCTATAGCGTAGCCCTCCCACCACCCC CGCATCG

73

CZ13

GGGCACACAACAACCGATGGTCCAGCCACCCGAATGACCAGCCCACCCGC CACCCCGCG

65

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Table 2

Aptasensor

ELISA



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Spiked concentration (ng/ml)

Amount found (ng/ml)

Recovery %

Soya milk

1.0

0.86

10

Mango Juice

Sample

Tomato

Plum 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Page 26 of 33

RSD%

Amount found (ng/ml)

Recovery %

RSD%

86

4.2

0.85

85

7.0

8.9

89

5.1

10.5

105

7.2

1.0

0.92

92.0

5.0

0.9

90

7.6

10

9.3

93.0

4.3

8.0

80

7.3

1.0

0.93

93.0

3.5

0.84

84

6.5

10

9.1

91

3.8

10.5

105

6.8

1.0

0.94

94

3.1

0.9

90

7.5

10

9.5

95

4.0

10.3

103

7.8

Scheme 1.


Page 27 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23


Fig. 1 27 ACS Paragon Plus Environment


16 16

A

14

14

Fluorescence intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Fluorscence Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 33

12 10 8 6 4

12 10 8 6 4 2

2 0

B

0 1

2

3

4

5

6

7

8

9

10

CZ13

Selection round

CZ6


CZ1

CZ2

CZ5

CZ12 library

Page 29 of 33

1 2 3 20 4 A 5 15 6 7 Kd= 65 nM 10 8 9 5 CZ13 10 11 0 12 0 100 200 300 13 DNA conc. (nM) 15 14 D 15 16 10 17 Kd= 73 nM 18 195 CZ6 20 21 0 22 0 100 200 300 23 DNA conc. (nM) 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Fig. 3

Fluorescence Intensity Fluoresce nce Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15

15

B

C

10

10

Kd= 72 nM 5

Kd= 250 nM 5

CZ2 0 400 0

100

CZ5

200

300

0 400 0

100

DNA conc. (nM) 20

400

F

15

15

Kd= 91 nM

10 5 0 400 0

300

DNA conc. (nM)

20

E

200

5

CZ1 100

200

Kd= 60 nM

10

300

DNA conc. (nM)


0 400 0

CZ12 100

200

DNA conc. (nM)

300

400


1 2 3 4 5 6

80

2100

A

40

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Bare Au Apamer/Au MCH/Aptamer/Au

1500

20

1200

0 -20

900 600

-40

300

-60 -0.2

B

1800 Bare Au Apamer/Au MCH/Aptamer/Au

-Z im (Ω )

60

Current (µA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

0

0.0

0.2

0.4

0.6

0

1000

E (V) vs Ag-AgCl

2000

3000

Zr (Ω)


4000

5000

Page 31 of 33

1 2

100 MCH/Aptamer/Au 0.01 ng/ml CBZ 0.1 ng/ml CBZ 1.0 ng/ml CBZ 10 ng/ml CBZ 100 ng/ml CBZ

2800

A

2400 2000

90 80 (R-Ro)/Ro %

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

-Zim (Ω )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1600 1200

B

70 60 50 40

800

30

400

20

0

10

0

1000 2000 3000 4000 5000 6000 7000

0.01

0.1

1

10

100

Log CBZ concentration (ng/ml)

Zr (Ω)


1000


1

80

(R-Ro)/Ro %

60

40

20

et hy l

C pa BZ r th ia athi m on et ho xa ca m rb ar yl tri flu ra lin lin ur on At Fe raz na ine m ip Is h op os ro tu ro n

0

m

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2


Page 32 of 33

Page 33 of 33

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1


TOC

2

3 4

5

6

7

8

9 33 ACS Paragon Plus Environment

Selection and Characterization of DNA Aptamers for Electrochemical

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