Recent advances in the development of electrochemical aptasensors for detection of heavy metals in food

Journal Pre-proof Recent advances in the development of electrochemical aptasensors for detection of heavy metals in food Liyuan Wang, Xianglian Peng, Hongjun Fu, Chao Huang, Yaping Li, Zhiming Liu PII:

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DOI:

https://doi.org/10.1016/j.bios.2019.111777

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BIOS 111777

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Biosensors and Bioelectronics

Received Date: 13 July 2019 Revised Date:

5 October 2019

Accepted Date: 8 October 2019

Please cite this article as: Wang, L., Peng, X., Fu, H., Huang, C., Li, Y., Liu, Z., Recent advances in the development of electrochemical aptasensors for detection of heavy metals in food, Biosensors and Bioelectronics (2019), doi: https://doi.org/10.1016/j.bios.2019.111777. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

1

Recent advances in the development of electrochemical aptasensors

2

for detection of heavy metals in food

3

Liyuan Wang1, Xianglian Peng*,1, Hongjun Fu1, Chao Huang2, Yaping Li1,

4

Zhiming Liu2,3

5 6 7

1 College of Food Science and Engineering, National Engineering Laboratory for

8

Deep Process of Rice and Byproducts, Central South University of Forestry and

9

Technology, Changsha 41004, China.

10

2 College of Environmental Science and Engineering, Central South University of

11

Forestry and Technology, Changsha 410004, China.

12

3 Department of Biology, Eastern New Mexico University, Portales, NM. 88130,

13

USA

14 15

* Corresponding author. Tel.: +86-731-85623240.

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E-mail address: [email protected] (X. Peng)

1

1

Highlights

2




years are described.

3 4







9 10 11

Various strategies for designing highly sensitive electrochemical aptasensors are proposed.

7 8

New methods for rapid detection with their advantages and disadvantages are reviewed.

5 6

New aptamers developed for heavy metals detection in food over the past five




Application of nanomaterials and electron mediators in electrochemical biosensors is discussed.

Abstract Heavy metal contamination in environment and food has attracted intensive

12

attention from the public since it poses serious threats to ecological system and human

13

health. Traditional detection methods for heavy metals such as atomic absorption

14

spectrometry have a fairly low detection limit, but the methods have many limitations

15

and disadvantages. Therefore, it is of significance to develop a rapid technology for

16

real-time and online detection of heavy metals. The electrochemical aptasensor-based

17

technology is promising in the detection of heavy metals with advantages of high

18

sensitivity, specificity, and accuracy. Although its development is rapid, more

19

researches should be carried out before this technology can be used for on-site

20

detection. In this review, the origin, basic principles and development of

21

electrochemical aptasensors are introduced. The applications of nanomaterials and

22

electrochemical aptasensors for the detection of heavy metals (mainly mercury, lead,

23

cadmium, and arsenic) are summarized. The research and application tendency of

24

electrochemical aptasensors for detection of heavy metals are prospected.

25 26 27

Keywords: Heavy metal; Detection; Nanomaterial; Electron mediator;

28

Electrochemical aptasensor

2

1

1. Introduction

2

Food is the basic condition for human survival, reproduction, and health

3

maintenance. Unsafe food causes various illnesses and even death (Sharma et al.,

4

2015). Food contamination is one of the main reasons accounting for food safety

5

issues. The common food contaminants include pesticides, toxins, heavy metals,

6

antibiotics and so on, and among these, heavy metals are a serious threat to food

7

safety (Lan et al., 2017).

8

Heavy metals, whose atomic weight is between 63.5 to 200.6, and with a specific

9

gravity greater than 5.0, include mercury (Hg), lead (Pb), cadmium (Cd), arsenic (As),

10

zinc (Zn), nickel (Ni), copper (Cu) and so on (Islam, et al., 2015; Zhang et al., 2015).

11

Mercury (Hg) is a toxic element that can be easily enriched in organisms through food

12

chains, not easy to discharge (Diop and Amara, 2016), and harmful to human health,

13

human nerves, kidneys, and liver in particular (Zhou et al., 2016; Vogel et al., 2016).

14

Although Hg is deposited mainly in its inorganic form it can convert into a more toxic

15

form, methyl mercury by anaerobic microbial activity (Matos et al., 2015). Methyl

16

mercury is a powerful neurotoxin that can pass through blood-brain barrier and

17

exhibit a serious negative impact on the human central nervous system. The Joint

18

Food and Agriculture Organization (FAO)/ World Health Organization (WHO) Expert

19

Committee on Food Additives (JECFA) has established strict standards for weekly

20

allowable intake of methyl mercury in the human body, i.e. not exceeding 1.6 µg·kg-1

21

(USEPA,1997). Lead is a toxic heavy metal and ranks the first among the six types of

22

heavy metal pollution (Silbergeld et al., 2010). Symptoms of acute lead poisoning in

23

humans include nausea, vomiting, paroxysmal abdominal cramps, sweat, headache,

24

convulsions, coma and circulation failure. Chronic poisoning is characterized by

25

muscle spasticity, blurred vision, unclear consciousness, and memory loss (Qasim et

26

al., 2014). The World Health Organization has established strict standards for the

27

weekly allowable intake of lead in the human body, i.e. not exceeding 0.025 mg·kg-1

28

(Joint FAO/WHO Expert Committee on Food Additives, & Organization, W. H. ,

29

2010). Cadmiumis a highly toxic heavy metal which has many toxic effects on the

30

kidneys, liver, nerves, and cardiovascular systems of the human body (Liu et al., 3

1

2015). Although its concentration is quite low in the natural environment it increases

2

in the human body through the food chain and causes humans to get ill under a

3

long-term exposure (Santos et al., 2016). In 1972, the United Nations Food and

4

Agriculture Organization (UNFAO) and the World Health Organization (WHO)

5

identified cadmium as a high priority contaminant in food. Among the 12 dangerous

6

toxic compounds of global significance proposed by the United Nations Environment

7

Programme in 1974, cadmium is listed as the first one (Varaksin et al., 2014). Arsenic

8

is also classified as heavy metal because its chemical properties are similar to heavy

9

metals (Zhan et al., 2016). There are inorganic arsenic and organic arsenic in food,

10

and the International Agency for Research on Cancer (IARC) has classified inorganic

11

arsenic and its compounds as primary carcinogens (Heinrich, 2003). For the above

12

reasons, the detection of heavy metals in food is very important.

13

Traditional detection methods for heavy metals include atomic absorption

14

spectrometry (AAS) (Zhou et al., 2016), atomic fluorescence spectrometry (AFS)

15

(Fernandez-Martinez et al., 2015 ), spectrophotometry (Wei-Hong et al., 2017),

16

inductively coupled plasma mass spectrometry (ICP-MS) (Bua et al., 2016), and

17

inductively coupled plasma atomic emission spectrometry (ICP-AES) (Zhao et al.,

18

2015). These methods can exactly determine heavy metals content in food, but the

19

limitations such as complicated sample pre-treatment steps, a relatively long detection

20

time, expensive equipment, and high operating costs, make them only applicable to

21

laboratory analysis and difficult to apply in field and other situations (Huang et al.,

22

2012). Therefore, rapid detection methods for heavy metal pollutants are needed.

23

Compared with traditional detection technology, rapid detection technology can

24

be qualitative or semi-quantitative for heavy metal contaminants, and are very suitable

25

for on-site detection of heavy metal pollution in food due to its advantages of

26

convenience, speediness and economy. The rapid detection technology has the

27

following advantages: 1) it works on site and can quickly and accurately analyze

28

various elements; 2) it needs small equipment and is easy to carry (Lin, et al., 2014;

29

Ma et al., 2015). Electrochemical biosensor is an analytical method for

30

electrochemical signal detection of biomolecules and has been widely used in the 4

1

fields of medicine, food safety testing, animal husbandry and veterinary,

2

environmental testing, and fermentation engineering since 1967 (Schaefer et al., 2003;

3

Luong et al., 2008). Table 1 lists some methods for rapid detection with their

4

advantages and disadvantages. Compared with other methods electrochemical

5

biosensors have the advantages that other rapid detection methods do not have.

6

Therefore, we can focus on improving the reproducibility and stability of

7

electrochemical biosensors in the future.

8

Table 1 Basic principles, advantages and disadvantages of the rapid detection

9

methods Detection methods

Detection principles

Advantages

Disadvantages

References

Enzyme analysis

Heavy metals can bind to the sulfhydryl or carbonyl groups that form the active center of the enzyme, thereby changing the structure and properties of the active center of the enzyme, thus establishing a quantitative relationship between the concentration of heavy metals and changes in the enzyme system. The heavy metal antibody immunoassay is based on the principle that the antibody and the corresponding antigen can be specifically recognized and combined with high affinity at a very low concentration, and the color reaction of the labeled secondary antibody and the primary antibody and the antigen complex to be tested is used to characterize and quantify heavy metals

High sensitivity

(a) It is difficult to detect a single heavy metal ion. (b) Only for water samples

Tolun et al. ( 2012)

Fast detection, high sensitivity and high selectivity

(a) Preparation of metal ion monoclonal antibodies is very difficult. (b) Polyclonal antibodies are difficult to meet the specific requirements for metal ions

Han et al. (2019)

Immunoassay

5

Biosensor

Test strip

1

Electrochemical biosensors use biological substances such as enzymes, antigens/antibodies, and DNA as molecular recognition elements and combine with electrochemical signal converters (electrodes) to convert biological signals acting on the target into identifiable electrical signals. The heavy metal content is detected by a color reaction of a heavy metal ion with a color developer.

Low detection limit, short response time, high sensitivity, specificity, simple operation, low manufacturing cost, and the ability to provide continuous real-time detection signals.

Poor anti-interferenc e ability

Arlett et al. (2011)

Cheap and simple

The detection limit is relatively high.

Wang et al. (2019).

According to the biomaterials fixed on the electrode the electrochemical

2

biosensors can be divided into electrochemical enzyme sensors, electrochemical cell

3

sensors, electrochemical immunosensors, electrochemical aptasensors, and

4

electrochemical microbial sensors (Dai et al., 2018). In recent years, aptamer has

5

become a research hot spot. Aptamer is typically a kind of artificial single-stranded

6

DNA or RNA, which generally is about 20~80 bases long, was first discovered in

7

1990 using a method called systematic evolution of ligands by exponential enrichment

8

(SELEX) in vitro (Alsager et al., 2014; Ellington et al., 1990; Tuerk et al., 1990). The

9

most remarkable features of aptamers are their high binding affinity and specificity.

10

Aptamers can bind to various targets such as heavy metal ions, proteins

11

(Amayagonzález et al., 2014), viruses (Park et al., 2014), and cancer cells (Sun et al.,

12

2016). Since aptamers are analogous to antibodies, they are also called the “forth

13

generation antibody” (Gopinath et al., 2016). In comparison with regular antibodies,

14

the aptamers have the following advantages: 1) Aptamers can be quickly synthesized

15

by chemical methods in vitro, but antibodies usually need to be synthesized by animal

16

or cell culture; 2) Aptamers are easy to modify, without affecting their biological

17

activities (Nutiu et al., 2010); 3) Non-immunogenic, non-toxic, and high tissue

6

1

permeability; 4) High stability. Aptamers consist of nucleotides with anti-denaturation,

2

which can withstand a wide range of pH and temperature, so they can be preserved for

3

a long period of time, while most of the antibodies are proteins which are prone to

4

denaturation (Hansen et al., 2006); 5) Wide range of target molecules (Zheng et al.,

5

2007; Pei et al., 2012).

6

When aptamers bind to a target molecule, they often lead to changes in the

7

structure and spatial conformation of the single-stranded nucleic acid. They can fold

8

into some specific secondary and tertiary structures by stacking of aromatic rings,

9

electrostatic, van der waals interactions and hydrogen bonding in the chain

10

(Stoltenburg et al., 2007; Zhan et al., 2016). For detection of Pb2+, most researches are

11

based on the aptamers to form G-quadruplex (Zhang and Wei, 2018). For detection of

12

Hg2+, T-T mismatches selectively capture Hg2+ to form T- Hg2+-T complex (Tan et al.,

13

2017). For detection of Cd2+, thymine (T) and guanine (G)- rich nonrepeating ssDNA

14

sequences are used to capture Cd2+ (Wu, et al., 2014). Based on the special structures,

15

the sensors can detect heavy metal ions accurately.

16

Based on the fast responsiveness, low cost of electrochemical analytical methods

17

and high affinity of aptamers, electrochemical aptasensors have attracted considerable

18

interests from many researchers. In this paper, the development and applications of

19

electrochemical aptasensor in the detection of heavy metals (mainly mercury, lead,

20

cadmium, and arsenic) are summarized. The prospect of electrochemical aptasensors

21

for detection of heavy metals in food is proposed.

22

2. Application of nanomaterials in Electrochemical Biosensors

23

Nanomaterials have many unique properties that differ from traditional materials,

24

such as small size, surface effect, quantum size, and macroscopic quantum tunneling

25

effect, and etc. Based on these characteristics nanomaterials are widely used to

26

modify electrodes. There are several reasons for introducing nanomaterials into

27

electrochemistry, including the occurrence of catalytic reactions, accelerated electron

28

transfer rates, immobilization of biomacromolecules, and use as switches for reactants.

29

In recent years, with the development of nanomaterial-modified electrodes and

30

biotechnology many nanomaterials can be combined with biomolecules to obtain 7

1

bioprobes for monitoring purposes. Therefore, combining the advantages of high

2

affinity and high specificity of nucleic acid aptamers with the above characteristics of

3

nanomaterials not only promotes the development of the two fields, but also has great

4

significance for achieving rapid and high sensitivity detection. Among the

5

nanomaterials used in electrochemical aptasensors for detection of heavy metal ions,

6

metallic nanomaterials and carbon nanomaterials are the most widely used (Gumpu et

7

al., 2015).

8

2.1 Application of metallic nanomaterials in electrochemical aptasensors

9

Noble metal nanoparticles are a special class of particles of intermediate

10

size (1~100 nm) between the atom cluster and micro-materials. They have

11

unique chemical and physical properties that differ from micro-materials, such

12

as small size and surface, excellent optical, electrical and catalytic properties

13

(Bradley et al., 2017). Among them, gold nanoparticles (AuNPs) and silver

14

nanoparticles (AgNPs) have been widely applied in many fields such as

15

development of catalysts and sensors (Sabela et al., 2017), as summarized in

16

Table 2.

17

2.1.1 Gold nanoparticles

18

As a kind of electrochemical modification of electrode materials, AuNPs

19

not only promote electron transfer between undetected object and electrode,

20

but also raise the sensitivity and catalysis. In addition, it can fix the living

21

biological cell and retain its biological activity (Abdelwahab, 2016). For

22

electrochemical aptasensor, AuNPs can be connected to aptamers by sulfydryls

23

and improve the electron transport rate and biocompatibility. An

24

electrochemical biosensor based on AuNPs-glutathione (GSH)/cysteine and

25

T-Hg2+-T structures was developed by Maatouk et al. in 2016. This AuNPs based

26

sensor increased the effective electrode surface area, accelerated the electron

27

transfer and achieved the rapid and cost-efficient analysis. The Hg 2+ was

28

detected and identified at trace level quantities with a detection limit of 50 pM.

29

Taghdisi et al. (2016) fabricated a selective and sensitive electrochemical aptasensor

30

for detection of Pb2+, which is based on gold nanoparticles (AuNPs) and the hairpin 8

1

structure of complementary strand of aptamer and thionine. Specifically, lead ions can

2

make complementary strand to form a hairpin structure, generating a weak

3

electrochemical signal. Conversely, a strong electrochemical signal will develop in the

4

absence of lead ions. The limit of detection for Pb2+ was measured as low as 312 pM.

5

In their another study, they developed an electrochemical aptasensing platform for

6

detection of Pb2+ as shown in Fig. 1 (Taghdisi et al., 2017). The detailed process is

7

based on the use of AuNPs, the enzyme exonuclease III, and the hairpin structure of

8

the complementary strand of an aptamer that prevents binding of AuNPs to the surface

9

of the sensor electrode in the presence of Pb2+. In the absence of Pb2+, AuNPs attach

10

to the surface of the gold electrode, and this leads to the generation of a strong

11

voltammetric signal. In the presence of Pb2+, AuNP does not bind to the electrode

12

surface due to the hairpin structure of the complementary strand, and this sensor has a

13

limit of detection (LOD) of 149 pM. Tian et al. (2015) attached the capture DNA to a

14

platinum electrode modified with three-dimensional ordered macroporous polyaniline,

15

and added gold nanoparticles with two probe DNAs to the solution. One probe DNA

16

binding to the capture DNA, and the other to Hg2+, then the hairpin is opened, and

17

hemin is added to form a G-tetraplex catalyzed H2O2, thus generating an electrical

18

current with a LOD of 87 fM.

19 20

Fig. 1 Schematic diagram of Pb2+ detection based on electrochemical aptasensors. In 9

1

the presence of Pb2+, the aptamer binds to Pb2+ and leaves the complementary strand,

2

which forms a hairpin structure that can protect the complementary strand against

3

digestion by Exo III and interaction with AuNPs, thus resulting in a weak

4

electrochemical signal (a). In the absence of Pb2+, the aptamer is hybridized with

5

complementary strand and disassembled the hairpin structure of complementary

6

strand. Then the aptamer is released after the addition of Exo III, and interacted with

7

AuNPs, resulting in a strong electrochemical signal (b). (Taghdisi et al., 2017).

8

Copyright 2017, SpringerLink.

9 10

In addition, AuNPs can be functionalized by DNA so that it contains a hairpin

11

probe DNA and a linear signal DNA with the function of a signal trigger. Zhang et al.

12

(2017) developed a facile and reusable approach to detect Hg 2+ based on the strong

13

T-Hg 2+ -T interaction with T-T mismatches. In this case, DNA1 is modified on

14

the Au electrode and DNA probe is complementary with DNA 1. In the

15

presence of Hg 2+ , the conformation of DNA probe changes from a line to a

16

hairpin, and less DNA probes are adsorbed to DNA 1. To improve the

17

sensitivity of this sensor, AuNPs are modified and DNA 3 is used to adsorb the

18

redundant DNA 1. He et al. (2018) designed an electrochemical biosensor based on

19

this structure, and it had a wide linearity of 0.35~3500 pM and a low Hg 2+ detection

20

limit of 0.21 pM (Fig. 2).

10

1 2

Fig.2 Schematic diagram of an electrochemical DNA biosensor for quantitative

3

monitoring of Hg (II) based on DNA functionalized Au nanoparticles and

4

nanocomposite modified electrodes. DNA-functionalized Au nanoparticle (DFNP)

5

include A hairpin probe DNA1(PD) with biotin and a linear signal DNA with

6

methylene blue-labeled, and nanocomposite and avidin were fixed on the electrode.

7

When Hg2+ and helper DNA (HD) were added, the T-T mismatches (PD and HD) can

8

capture Hg2+ to form T- Hg2+-T complex, and the hairpin structure of PD was opened

9

and makes biotin to be recognized by avidin, which results in DFNP being brought

10

onto the electrode and generating a strong DPV signal. When only HD was added, the

11

hairpin structure of PD cannot be opened and the distinction between biotin and

12

avidin would not be found, which results in a weak DPV signal. (He et al., 2018)

13

2.1.2 Sliver nanoparticles

14

From the above it can be seen that AuNPs have many unique properties. But

15

there are many problems, such as how to make AuNPs not to agglomerate, how

16

to prevent non-specific adsorption of AuNPs onto biomolecules. Compared

17

with AuNPs, application of AgNPs in sensors is not widespread. Ebrahimi et al.

18

(2015) designed an electrochemical biosensor based on a stable T-Hg 2+ -T

19

structure. In this system, carbon paste electrode was modified with AgNPs in 11

1

order to improve the efficiency of biosensor. The linear range of this biosensor was

2

9.0×10-11~1.0×10-9 M, with a detection limit of 3.08×10-11 M. Alex et al. (2017)

3

developed a protocol for the detection of ultra-trace levels of Hg2+ in aqueous

4

solutions by linear sweep voltammetry (LSV) with AgNPs modified glassy carbon

5

electrode. This was achieved by detecting the change in the silver stripping

6

peak with Hg 2+ concentration resulting from the galvanic displacement of

7

silver by mercury: Ag(np) + 1/2Hg 2+ (aq) → Ag + (aq) + 1/2Hg(l). The

8

calibration curve was linear from 100.0 pM to10.0 nM. The limit of detection

9

under the optimal conditions was 28 pM based on 3σ. Xie et al. (2018) designed a

10

biosensor based on the highly effective decomposition of a branched DNA polymer

11

and target-transition recycling amplification used for detection of Pb2+. DNA polymer

12

was fixed on the electrode surface and AgNPs were used to generate a high initial

13

current signal. The molecule then triggers T-1 and T-2, which are produced by

14

recirculating the target conversion, which can disassemble the backbone and

15

side chains of the branched DNA polymer, significantly reducing the current.

16

Compared with the traditional methods, this sensor has an excellent

17

performance with a LOD of 0.24 pM.

18

2.1.3 Metal nanocomposites

19

Different metal materials or metal composite materials such as metal nanorods,

20

metal core-shell structures, irregular metal nanostructures and spherical metal

21

nanomaterials are used in the construction of sensors (Alkilany et al., 2012). Zhao et

22

al. (2017) synthesized Au@Ag core-shell nanoparticles and used for the construction

23

of electrochemical aptasensors by square wave voltammetry (SWV) (Fig. 3 and Fig.

24

4). The results showed that the sensor achieved the superior reversibility,

25

stability, repeatability, and selectivity for the detection of Hg2+ , and had a

26

linear detection range of 2~20 µg/L, with a detection limit of 0.006 µg/L. In

27

this detection system, Au@Ag core-shell nanoparticles enlarge the specific surface

28

area and amplify the ability of catalysis. In another study, Xu and colleagues

29

developed an aptasensor, which used AgPt nanoparticles (AgPtNPs) as

30

electrochemical probes and signal enhancers. The aptasensor had a wide linear range 12

1

of 0.1 pM to 100 nM with the LOD of 0.032 pM (Xu et al., 2017). Ding et al. (2018)

2

used gold nano/polypyrrole-modified screen-printed electrodes for the determination

3

of Pb2+. Gold nano/polypyrrole improved the surface area of screen-printed electrodes

4

and amplified the current. The detection limit of this method was 0.36 nM.

5 6

Fig. 3 (A) Construction process of DNA2-Au@Ag-DNAzyme conjugate; Au@Ag

7

nanoparticles were modified by DNA2 and DNAzyme. (B) Manufacturing process

8

and measurement principle of electrochemical aptamer biosensor. When

9

DNA2-Au@Ag-DNAzyme and Hg2+ are added, the double-strand DNA structure is

10

formed based on T-Hg2+-T, which results in DNA2-Au@Ag-DNAzyme being

11

brought onto the electrode surface and generating a strong SWV signal. (Zhao et al.,

12

2017)

13 14

Fig.4 (A) The SWV curves toward different Hg2+ concentration. (B) Calibration curve

15

of the biosensor between current and concentrations of Hg2+ (0, 0.02, 0.2, 0.5, 1, 2, 4,

16

10, 20 µg L-1). The current intensity increase gradually with the increase of Hg2+

13

1 2

concentration within limits. (Zhao et al., 2017) Table 2 Determination of heavy metals by electrochemical aptasensor based on metallic nanomaterials.

3

Relative Heavy

Electrode

Limit of

Linear

standard

detection

range

deviations

Technique metal ions

material

References (RSDs)

Hg2+

50 pM~1 AuNPs

DPV EIS

50 pM

Maatouk et 5.0% al. (2016)

µM Pb2+

Taghdisi et AuNPs

DPV

312 pM

0.6~50 nM al. (2016)

Pb2+

Hg

2+

0.7~300 AuNPs

AuNPs

DPV

DPV

149 pM

Taghdisi et 5.3%

nM

al. (2017)

10-11~10-5

Tian et al.

M

(2015)

8.7 fM 0.35

Hg2+

He et al. AuNPs

DPV

0.21 pM

pM~3500 (2018) pM Zhang et al.

Hg2+

CV AuNPs

0.1~200 0.05 nM

EIS

Hg

2+

Pb2+

AgNPs

2.7%

3.08×10-11

9.0×10-11 ~

Ebrahimi et

M

1.0×10-9 M

al. (2015)

DPV

1 pM~100 AgNPs

LSV

(2017)

nM

0.24 pM

Xie et al. 4.3%

nM

(2018)

Au@Ag Hg2+

core-shell

Zhao et al. SWV

0.006 µg/L

nanoparticl

2~20 µg/L (2015)

es

14

Pb2+

0.1 pM AgPtNPs

DPV

0.032 pM

Xu et al. 4.9%~6.5%

~100 nM

(2017)

gold@poly Pb2+

Ding et al. pyrrole

DPV

0.36 nM

0.5~10 nM

4.36% (2018)

composites 1 2

2.2 Application of carbon nanomaterials in electrochemical aptasensors Carbon nanomaterials include fullerenes, carbon nanotubes (CNTs), graphene (GR)

3

and its derivatives, graphene oxide (GO), nanodiamonds (NDs), and carbon-based

4

quantum dots (CQDs) (Patel et al., 2019). Among them, GR and CNTs are the most

5

widely used carbon nanomaterials. The surface of carbon nanomaterials is easily

6

modified by non-covalent or covalent methods. Non-covalent modification relies on

7

π-π stacking on the surface of the modifier and carbon nanomaterial (Wang et al.,

8

2016). The covalent structure exploits the π-π stacking based on sp2-hybridized

9

carbons. Therefore, carbon nanomaterials have a high application potential in

10

electroanalytical chemistry, as summarized in Table 3.

11

2.2.1 Graphene

12

Graphene (GR) has been widely used as electrode material due to its excellent

13

properties such as large specific surface area, high electrochemical stability, fast

14

electron transfer rate and high conductivity (Alwarappan et al., 2009; Stoller et al.,

15

2008; Wang et al., 2014). Hence, the aptamer-based biosensor can be constructed by

16

immobilizing the aptamer on GR to improve its performance. The remarkable

17

property of GR lies in its excellent electrode transfer performance. Gao and

18

colleagues designed an electrochemical aptasensor for detection of Pb2+ by absorbing

19

it on the electrode surface based on the π–π stacking interactions between GR and

20

aptamer (Gao et al., 2014). Analysis by electrochemical impedance spectroscopy

21

shows that GR can reduce the electron transfer resistance at the electrode interface,

22

that is, it promotes the transfer of electrons and improves the sensitivity of the sensor.

23

Pb2+ can convert aptamer to a stable G-quadruplex structure and cause changes in

24

electrical signals. Under optimal experimental conditions, the attenuation of peak

15

1

currents showed a linear relationship with the logarithm of Pb2+ concentrations over

2

the range of 5.0 ×10-10 to 5.0 × 10-8 M. The detection limit was 6.0 × 10-11 M. In

3

another study, Gao and colleagues constructed a label-free and highly sensitive

4

electrochemical aptasensor for Pb2+ by using thionine (TH) as the signaling molecule

5

and graphene (GR) as the signal-enhancing platform (Fig. 5) (Gao et al., 2016).

6

Through cyclic voltammetry analysis the redox signal was significantly smaller from

7

the electrode assembled with GR than that from the electrode without GR. This

8

indicates that GR plays an important role in the electrochemical response of the sensor.

9

As a result, the attenuation of peak currents increased with Pb2+ concentration over a

10

linear range of 1.6×10-13 to 1.6×10-10 M. The detection limit was 3.2×10-14 M. There

11

are several merits for the aptasensor, such as good regenerability, excellent selectivity,

12

and reproducibility.

13 14

Fig. 5 A schematic of an electrochemical Pb2+ aptamer sensor based on the

15

progressive assembly of GR and TH on the surface of an aptamer-modified electrode.

16

In the presence of Pb2+, the free lead (II) specific aptamer (LSA) is changed to the

17

folded G-quadruplex structure through its specific interaction with Pb2+. The affinity

18

of GR with LSA is significantly reduced, and as a result, the complex of GR with the

19

electroactive TH is liberated from the sensing interface, resulting in the reduction of

20

the redox signal of the sensor. (Gao et al., 2016)

21

2.2.2 Graphene oxide

22

Reduced graphene oxide (RGO) is a newly two-dimensional planar structure 16

1

carbon materials that were discovered in recent years (Li et al., 2012). RGO not only

2

has sheet structure of GR to enlarging specific surface area, and reduces some

3

oxygen-containing functional groups such as -OH, -C=O, etc., to increase the electron

4

transfer rate and facilitate adhesion of other metal particles to the surface. These

5

excellent properties make RGO widely used in the field of sensors (Liu et al., 2014).

6

Zang et al. (2014) designed a “signal-on” photoelectrochemical sensing strategy for

7

detection of Pb2+ based on the amplified effect of RGO. Electrochemical impedance

8

analysis of the bare electrode and the electrode modified with RGO showed that the

9

latter had a lower ion–electron charge transfer resistance at the interfaces (RCT)

10

compared to the former. This was mainly due to the excellent electron transfer

11

performance of RGO. The aptasensor showed a linear relationship between

12

photocurrent variation and the logarithm of Pb2+ concentrations in the range of 0.1–50

13

nM with a detection limit of 0.05 nM. Zhang et al. (2015) developed an

14

electrochemical biosensor for detection of Hg2+ based on thymine-Hg2+-thymine

15

(T-Hg2+-T) complex and the remarkable difference in the affinity of graphene to

16

double stranded DNA (ds-DNA) and single stranded DNA (ss-DNA). The large

17

specific surface area of GR promotes the transfer of electrons. The linear range of this

18

biosensor was 5.0×10-10 M to 1.0×10-7 M, with a detection limit of 1.2×10-10 M. Wang

19

et al. (2018) developed a novel sensor by fabricating a system of specific aptamers

20

and RGO/graphite-carbon nitride (G-C3N4) (GCN).The biosensor showed high

21

stability, specificity, sensitivity, and reproducibility for detection of Cd2+. The linear

22

response range of the aptasensor was 1 nM to 1 µM with a limit of detection of 0.337

23

nM.

24

2.2.3 Carbon nanotubes

25

Carbon nanotubes (CNTs) are an excellent carrier material with low electrical

26

resistance, remarkable mechanical property, and high conductivity (Zhu et al., 2018).

27

Lian et al. (2014) developed an electrochemical sensor based on the interaction of

28

Pb2+ with nucleic acid via phosphate groups and nucleic bases of the deoxyribonucleic

29

acid structure. In this system, as a material for modifying the electrode, metallic

30

single-walled carbon nanotubes are wrapped by DNA, which not only fix the DNA to 17

1

the electrode, but also serve as a powerful amplifier to improve the detection limit and

2

sensitivity of sensor. The limit of detection for Pb2+ was measured as low as 4.1×10-10

3

M.

4

2.2.3 Carbon nanocomposites

5

The performance of individual carbon nanomaterials is relatively simple, while

6

the performance of metal/carbon nanocomposites is diverse. At present, there are

7

many reports on the use of nanocomposite-modified electrochemical biosensors for

8

detection of heavy metals. In the above, we mentioned some excellent properties of

9

CNTs. However, it is worth noting that CNTs formed the net structure in the

10

interlaced form on electrodes due to their structural flexibility. The structures can

11

hinder the electron transport and reduce the electron transfer rate. Thus, a good

12

electrical conductivity of other materials is necessary to connect them and form a

13

smooth electronic transmissions network (Sun et al., 2016). For this reason, Zhu and

14

colleagues developed an electrochemical sensor based on carboxylic acid group

15

functionalized multiwalled carbon nanotubes (MWNTs-COOH) and direct

16

electrodeposited gold nanoparticles (GNPs) (Zhu et al., 2014). Scanning electron

17

microscopy (SEM) analysis showed that the spatial structure of MWCNTs/GNPs

18

provided the binding sites and spaces for subsequent reactions. Analysis of the

19

electrical resistance of MWCNTs/GNPs showed that with the modification of GNP,

20

almost linear lines were observed, which means there is an increase in the electron

21

transfer rate. This sensor could detect Pb2+ in a range of 0.01~50 pM with the LOD of

22

0.0043 pM. Hai et al. (2014) developed an electrochemiluminescence (ECL) for

23

detection of Pb2+ ions. The aptamers labeled with quantum dots (QDs) were

24

immobilized on the surface of graphene and AuNPs modified indium tin oxide (ITO).

25

In the presence of Pb2+, the ECL signal was quite strong because Pb2+ induced the

26

stem-loop of the aptamer to form a G-quadruplex and the amino group at the 3′-end

27

was exposed and could covalently link to a carboxyl group on the surface of the CdTe.

28

In the absence of Pb2+, the hairpin capture probe was unable to interact with QDs due

29

to its steric hindrance. Using electrochemical impedance spectroscopy (EIS)

30

technique, the linear range was reported to be from 10 pM to 1 nM with the LOD of 18

1

3.8 pM. For this aptasensor, the gold nanoparticles can prevent the accumulation and

2

enhance the electronic conductivity of graphene. Zhang et al. (2015) designed an

3

electrochemical biosensor for detection of Hg2+ (Fig. 6), which includes three ssDNA

4

(P1, P2, P3). Among them, P1 was modified by GR and nanoAu, then placed in Hg2+

5

solution containing P2, followed by contacting with P3 labeled with nanoAu and

6

methyl blue (MB-nanoAu-P3s) for hybridization. In the absence of Hg2+, P1 and P2

7

were unable to form double-stranded DNA on the surface of electrode. At the same

8

time, P3 labeled with MB showed no current response. In the presence of Hg2+, a T-T

9

mismatched dsDNA would occur between P1 and P2 on the electrode surface due to

10

the formation of T-Hg2+-T structure. Because of a partial sequence complementation

11

in P1 and P2, MB-nanoAu-P3s and P2 could form a dsDNA by hybridization, leading

12

to the changes of electrochemical signals. The linear range of the sensor for detecting

13

Hg2+ was from 1.0 aM~100.0 nM with a detection limit of 0.001 aM. Lei et al. (2015)

14

constructed a label-free ratiometric aptasensor for detection of Pb2+ using a glassy

15

carbon electrode with gold-nanoparticles-functionalized fullerene nanocomposites

16

(AuNPs@nano-C60) bounded thiol-modified assistant probes (APs). The resultant

17

electrode was hybridized with Pb2+ aptamer (CPs) to generate DNA duplexes. Using

18

electrochemiluminescence (ECL) technique, the limit of detection was 3.5 × 10−13 M.

19

Chang et al. (2015) used the graphene oxide on the gold interdigitated electrode,

20

and then modified the gold nanoparticles and the T-rich DNA. After adding Hg2+, the

21

DNA formed a T-Hg2+-T structure. Different concentrations of Hg2+ made the source

22

to leak. The current between the poles changed, and online monitoring of Hg2+ was

23

achieved. The detection limit of the sensor was 1.0 nM. Luo and colleagues

24

constructed a sandwich-type aptasensor based on Fe3O4/rGO nanocomposites as

25

signal amplifiers (Luo et al., 2017). The Hg2+ aptasensor showed a wide linear range

26

from 0.1~100 nM with a low detection limit of 30 pM. Compared with the traditional

27

metal nanoparticles (NPs)-based method, the aptasensors not only reduced the cost,

28

but also improved the sensitivity of the heavy metal detection.

19

1 2

Fig. 6 Schematic diagram of the sensing principle of mercury detection. In the

3

presence of Hg2+, a T-T mismatched dsDNA forms between P1 and P2 on the

4

electrode surface due to the formation of T-Hg2+-T structure. Because of a partial

5

sequence complementation in P1 and P2, MB-nanoAu-P3s and P2 form a dsDNA by

6

hybridization, leading to the changes of electrochemical signals. (Zhang, et al., 2015)

7

Polymer/Carbon nanomaterials have raised many researchers’ interests.

8

Conducting polymers (CPs) generally consist of the polymer chain and one-valence

9

anion or cation of non-bonding chains (Zhou et al., 2014). CPs not only contain the

10

intrinsic properties of polymer, but also include the properties of conductors and

11

semiconductors. They have been widely used in many fields due to their remarkable

12

properties such as fast electron transfer rate, high sensitivity and excellent

13

biocompatibility (Li et al., 2014). Yang et al. (2015) synthesized three-dimensional

14

reduced graphene oxide and polyaniline (3D-rGO@PANI) and applied it to the

15

modification of electrode for detection of Hg2+. Compared with pure PANI,

16

3D-rGO@PANI exhibited enhanced conductivity and high stability. This method had

17

a detection limit of 0.035 nM and a linear range from 0.1 nM to 100 nM. Peng et al.

18

(2016) prepared a nanocomposite consisting of three-dimensional reduced graphene

19

oxide (3D-rGO) and plasma-polymerized propargylamine (3D-rGO@PpPG) and used 20

1

it to detect Hg2+. It showed high sensitivity and selectivity. Liu and colleagues

2

developed an electrochemical DNA sensor based on immobilizing the thymine

3

(T)-rich DNA on the surface of the electrode modified by Cu2O@NCs and measuring

4

changes in the surface properties (Liu et al., 2015). The detection limit was 0.15 nM.

5

Table 3 Determination of heavy metals by electrochemical aptasensor based on

6

carbon nanomaterials Heavy metal ions

Electrode material

Technique

Limit of detection

5.0×10-10～ 5.0 ×10-8 M 1.6×10-13~1. 6×10-10 M 5.0×10-10 ~1.0×10-7 M

2+

GR

DPV

6.0×10

Pb2+

GR

DPV

3.2×10-14 M

Hg2+

GR

DPV

1.2×10-10 M

Pb2+

RGO

Photoelectro chemistry

0.05 nM

0.1~50 nM

4.6%

Cd2+

RGO

DPASV

0.337 nM

1 nM~1µM

2.82%

Pb2+

RGO

DPV

0.51 fM

10-15~10-9 M

Hg2+

RGO

DPV

0.16 fM

Pb2+

SWNTs

DPV

4.1×10-10 M

Pb2+

SWNTs

FET

Pb2+

MWCNTs/G NPs

DPV

0.0043 pM

Pb2+

GR/AuNPs

ECL

3.8 pM

Hg2+

GR/AuNPs

DPV

0.01 aM

Hg2+

GR/AuNPs

FET

1 nM

1~50 nM

17.20%

Hg2+

Fe3O4/rGO

DPV

30 pM

0.1~100 nM

6.1%

Pb2+

AuNPs@na

ECL

3.5 × 10−13

1.0 × 10−12

4.12%

Pb

-11

Linear range

Relative standard deviations (RSDs)

M

0.39 ng/L.

21

0.1 fM~100 nM 1.0×10-9 ~1.0×10-8 M 1 ng/L ~ 100 µg/L 0.01~50 pM 10 pM~1 nM 1.0 aM~100.0 nM

3.1% 7.9% 1.6%

＜5%

References

Gao et al. (2014) Gao et al. (2016) Zhang et al. (2015) Zang et al. (2014) Wang et al. (2018) Yu et al. (2019) Yu et al. (2019)_ Lian et al. (2014) Wang et al. (2018)

4.8%

Zhu et al. (2014)

0.96%

Hai, et al. (2014)

4.49%

Zhang et al. (2015) Chang et al. (2015) Luo et al. (2017) Lei et al.

no-C60 Hg2+ Hg2+

3D-rGO@P ANI 3D-rGO@P pPG)

M

M ~1.0 × 10−7 M

(2015)

EIS

0.035 nM

0.1~100 nM

4.5%

DPV

0.02 nM

0.1~200 nM

<10%

Yang et al. (2015) Peng et al. (2016)

1 2 3

3. Application of electron mediator in electrochemical biosensors Electron mediator exhibits unique electrochemical activity, such as excellent

4

electron transfer ability and improved electron response (Jalalian et al., 2018).

5

Representative electrochemical aptasensors for various heavy metals based on various

6

electron mediators including their detection limit and linear range are listed in Table 4.

7

Lin et al. (2017) designed a novel strategy for selective and sensitive amperometric

8

detection of Pb2+ based on the formation of G-quadruplex structure with the LOD of

9

1.2×10-10 M. The ferrocene (Fc) is used as an electron mediator and can be labeled

10

onto the thiol-aptamer. The conformational change from coil-like to quadruplex

11

structure upon association of aptamer with Pb2+ induces a significant increase in the

12

Fc anodic current peak, thus facilitating a “switch on” detection. A “signal-on”

13

electrochemical sensor has been reported for detection of Hg2+ based on alternating

14

current voltammetry (ACV) measurements (Cui, et al., 2015). The three-way junction

15

(TWJ) consists of a capture probe that self-assembles on the gold electrode surface by

16

the S-Au bond. The signal probe is labeled with Fc and contains a single T-T

17

mismatch to capture the probe, and the auxiliary probe is used to form DNA-TWJ in

18

the presence of Hg2+. This process makes the Fc close to the electrode for the rapid

19

electron transfer, thereby increasing the oxidation current. A detection limit of 0.005

20

nM Hg2+ was obtained.

22

1 2

Fig.7 Schematic of a dual functional electrochemical switch sensor for monitoring

3

Hg2+ and melamine. A thiol-modified methylene blue (MB)-labeled thymine (T)-rich

4

SH-MB-DNA probe self-assembles on a gold sensing electrode. When Hg2+ added,

5

SH-MB-DNA is folded and brings the MB labels close to the electrode surface, which

6

results in an enhanced current response. Later, when melamine added, Hg2+ is released

7

from Hg2+-folded SH-MB-DNA and forms a more stable Hg2+- melamine

8

multi-nitrogen heterocyclic ring special structures, which results in a weak current

9

response. (Jiang et al., 2015)

10

Additionally, some organic dyes can also be utilized as electron mediator, such as

11

phenothiazine dyes (Thionine, meldola blue), methylene blue (MB), and neutral red

12

(NR). The advantages of the organic dyes as the electron mediator lie in their good

13

sensitivity, quick current response and facile preparation (Du et al., 2016). Xiong et al.

14

(2015) developed a simple, selective and reusable electrochemical biosensor for

15

detection of Hg2+ based on T-Hg2+-T structure. The thiolated methylene blue (MB)

16

modified T-rich hairpin DNA capture probe (MB-P) was first self-assembled on the

17

gold electrode surface by Au-S bonds. In the presence of Hg2+, a ferrocene

18

(Fc)-labeled T-rich DNA probe (Fc-P) mediated the coordination of T-Hg2+-T base

19

pairs by hybridization of Hg2+ with MB-P. Consequently, when the hairpin MB-P was 23

1

opened, the MB tags were away from the gold electrode and the Fc tags were closed

2

to the gold electrode. These conformational changes led to a decrease of the oxidation

3

peak current of MB with an increase of that of Fc (Fig. 7) (Jiang et al., 2015). The

4

development of a dual functional electrochemical switching sensor with the ability to

5

detect Hg2+ and melamine is described. A thiol-modified methylene blue

6

(MB)-labeled thymine (T)-rich SH-MB-DNA probe self-assembles on a gold sensing

7

electrode, and the detection limit is 8.2 nM. Li et al. (2015) assembled a

8

double-stranded DNA with NH2 and thiopurine on both ends to assemble the

9

cyclodextrin on the surface of the gold electrode. After Hg2+ is added, the

10

double-stranded DNA unwinds, the sulfur-containing chain enriched with T bases

11

interacts together to form a T-Hg2+-T structure. The T-Hg2+-T structure bends the

12

chain to shield the gap between the cyclodextrins, so that the surface resistance of the

13

probe entering the electrode becomes large, and the electrical signal decreases. The

14

sensor has a detection limit of 5 fM. Vega-Figueroa et al. developed an

15

arsenic-specific aptamer (ArsSApt)-based electrochemical method to study the

16

interfacial properties of an arsenic (As3+) sensor. The self-assemled ArsSApt layer

17

binding to As3+ on a gold substrate causes conformational changes at the interfacial

18

layer, then resulting in a detectable change in the impedimetric signal. The signal is

19

linearly correlated with As3+ concentration with a range of 0.05–10 ppm and a

20

detection limit of 0.8 µM (Vega-Figueroa et al. 2018).

24

1 2

Table 4 Determination of heavy metals by electrochemical aptasensor based on electron mediator

Heavy metal

Electron

ions

mediator

Hg2+

As3+

Technology

SWV

MB

SWV

TH

DPV

[Ru(NH3)6]3+

+

Limit of

Linear range

References

10 pM ~50

Hong et al.

nM

(2017)

detection

MB

[Ru(NH3)6]

Methods

T-Hg2+-T Y-shaped

1.6 pM 0.094 nM

1 nM~5µM

T-Hg2+-T

0.33 nM

1~200 nM

SWV

T-Hg2+-T

1 pM

0.01~500 nM

DPV

T-Hg2+-T

0.12 pM

DNA

3

0.2 pM~ 35

(2016) Gan et al. (2015) Bao et al. (2015)

nM

(2015)

SWV

T-Hg2+-T

8.7 × 10-11 M

MB

SWV

T-Hg2+-T

0.1 nM

0.2~100 nM

Ethyl green

DPV

T-Hg2+-T

0.0308 nM

0.09~1.0 nM

EIS

T-Hg2+-T

0.15 nM

1~100 nM

0.8 µM

0.05~10 ppm

25

Wu et al.

Chen et al.

MB

3

(2016)

nM 0 pM~500

EIS

Jia et al.

Tortolini et al. (2015) Ebrahimi et al.

(2015)

Liu et al. (2015) Vega-Figuero aet al. (2018)

1

4. Conclusions and outlook

2

Since the aptamer is easy to prepare, modify, and can bind to a wide range of

3

objects, it has attracted intense attention of researchers. Electrochemical technology is

4

playing an increasingly important role in the field of the aptamer biosensor. The

5

appearance of new materials has brought the development of electrochemical

6

aptasensors into a new stage. They provide not only excellent nano-sensitive materials,

7

but also many new methods for sensor design. Nanoparticles play an important role in

8

the field of electroanalysis, and their use in electrochemical aptamer sensors will

9

continue to expand. Graphene exhibits a unique advantage in electron transfer, which

10 11

will become one of the key electrode modification materials. This review presented an overview of a variety of aptamer-based approaches of

12

modification and combination of targets that can be applied to the detection of Pb2+、

13

Hg2+、Cd2+、As3+ in biological samples. We have seen the advantages of lower LOD

14

and high sensitivity of electrochemical aptasensor from this paper, but most

15

biosensors are not yet available for application. The main reasons are as follows: (1)

16

Most of the aptamers reported are only for one analyte, the aptamer used for

17

simultaneous, efficient, rapid and accurate detection of multiple analytes should be

18

focused in the future studies. (2) Although the performance of the sensors is good

19

under the laboratory conditions, the aptamers can be destroyed and degraded easily in

20

relatively harsh conditions. Therefore, it is necessary to strengthen the stability of the

21

sensors in the future. (3) The pretreatment of food samples is cumbersome, which is

22

always the problem that researchers need to solve. (4) The cost for testing at present is

23

high under the laboratory conditions. As research continues to deepen,

24

electrochemical aptamer sensors utilizing nanotechnology will play a greater role in

25

detection and are also expected to be used in many other fields.

26

Acknowledgements

27

We are grateful to the Hunan Provincial Natural Science Foundation Youth Fund

28

Project (Grant No. 2017JJ3521) and the National Natural Science Foundation of

29

China (Grant No. 51809293) for supporting this work.

26

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CRediT Author Statement Liyuan Wang is responsible for puting forward ideas and writing the paper. Xianglian Peng and Hongjun Fu are offer guidance in the processof writing the paper Chao Huang and Zhiming Liu are responsible for revising the paper. Yaping Li is responsible for collating literatures.

Declaration of interests


The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: