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Graphene and Graphene Oxide Based Biosensor For DNA Detection Chen Yao E-mail: [email protected] Contents 1 Introduction 2 2 Graphene Family Material 3 3 Material Properties Related to DNA Detection 5 3.1 Surface area .................................... 5 3.2 Surface Chemistry ................................ 6 3.3 Electrochemistry ................................. 8 4 Graphene-Based Sensors 9 4.1 Electrochemical DNA Sensors .......................... 9 4.2 Electronic Sensors ................................ 11 4.3 Optical DNA Biosensors ............................. 13 5 Conclusions and Outlook 15 Abstract 1
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Page 1: Graphene and Graphene Oxide Based Biosensor For DNA Detection · 2019-01-29 · Graphene and Graphene Oxide Based Biosensor ... Graphene oxide(GO) is a highly chemically modified

Graphene and Graphene Oxide Based Biosensor

For DNA Detection

Chen Yao

E-mail: [email protected]

Contents

1 Introduction 2

2 Graphene Family Material 3

3 Material Properties Related to DNA Detection 5

3.1 Surface area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3.2 Surface Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.3 Electrochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4 Graphene-Based Sensors 9

4.1 Electrochemical DNA Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . 9

4.2 Electronic Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

4.3 Optical DNA Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

5 Conclusions and Outlook 15

Abstract

1

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Owing their extraordinary electrochemical, electronic and optical properites, Graphene-

based materials show huge potential to fabricate the biosensor to detect DNA(cDNA,

ssDNA, dsDNA). There are three main types of graphene DNA sensors, electrochem-

ical, electronic and optical. In this review, I will survey properties and applications

of variety noval graphene DNA detecting sensors and o↵er insights on the underlying

DNA detection mechenisms.

1 Introduction

High selective, rapid, fast responding and cost e↵ective DNA detection biosensor is expected

in disease diagnosis and treatment, cancer detection and gene mutation in recent years.Using

nanomaterial to fabricate the biosensor drove the scientists attention .1–3 Graphene-family

material (especially for graphene and graphene oxide) is really interesting in this type biosen-

sor assembling due to its extremely high surface area, exceptional electronic and electro-

chemical properties.4 In this article, I aim to survey properties and applications of variety

noval graphene DNA detecting sensors and o↵er insights on the underlying DNA detection

mechenisms.

As a famous type of two-dimensional nanomaterial, graphene was first discovered in 2004

and won the Nobel Prize in 2010. The typical unique nanostrucuture is two-dimensional

carbon sheet with honeycomb-like arrangement of atoms, shown in figure 1. Due to this

special nanostructure, graphene owes the attractive electronic and electrochemical proper-

ties. It shows impressive carrier mobiliity and carrier density at room temperature.5 The

electrochemical potential of graphene is 2.5 V in 0.1 PBS which is higher than most of the

carbon based material.6 All those promising properties drove the attention of the scientists

and companies on the work of developing electronic, electrochemical and optical DNA de-

tection sensor based graphene-family material. How the DNA interacting with the graphene

is also vary important in DNA detection. In this review, I will summarize the varies DNA

detection sensors based on graphene family material and the involved interaction between

2

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DNA sequences and graphene material. Some methods of the functionalization of graphene

material could improving the performance of the sensors, which also will be mention in this

article.

2 Graphene Family Material

As developing in the past decade, there are plenty of graphene-family material having been

fabricated for variety applications. Graphene, few-layer-graphene (FLG), graphene oxide

(GO) and reuduced graphene oxide (rGO) are most advanced for bio-detection application.

Single layer graphene was first isolated from graphite using special adhesive tapel in 2004

by a research group from Mechester University led by Geim.7 Then there are two main tech-

nologies becoming vary popular in graphene production, repeated mechanical exfoliation of

graphite flakes and growth by chemical vapor deposition.8 The chemical process to fabricat-

ing graphene shows better biological performance might due to larger substrate areas than the

other ways. The typical graphene nanostructure is already mentioned above.(shown in figure

1) While there are still some problems for pristine graphene that it can not be suspended

in aqueous solution vary well, which is important in biological applications. Therefore, the

other types of the graphene-family material are more interesting for DNA detection which

will discuss in later sections. Few-layer-graphene was found as byproduct of the fabrication

for pristine graphene. It is general that these ultrathin graphene film, 1–10 layers, on crys-

talline transition metals substrates might introduce some contamination into the products

might showing unexpected thermal expansion or electronic behaviors.8

Graphene oxide(GO) is a highly chemically modified graphene which containing oxygen

functional groups and the chemical atom analysis shows the ratio of carbon to oxygen be

around three to one.1 It is one of the most admired graphene nano-material for biological

applications in recent years because it could be made as a stable, homogeneous GO aqueous

suspension, which is really important in DNA detection field.9 Just like in figure 1, as a

3

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1.jpg

Figure 1: Example members of the graphene nanomaterial family and selected propertiesrelevant to colloidal behavior and biological interactions. Graphene oxide sketch adaptedwith permission.8 Copyright 2009 C. Hamilton.

highly oxidized product of graphene, the surface structure of GO is consists of a single

layer graphene with oxygen functional groups such as carboxylate acid, epoxy and alcohol

groups on the periphery and the carbon to oxygen ratio have been analyzed to be around

three to one. Those functional groups provide pH dependent nagative charge and colloidal

stability.10–12

There are also unmodified graphenic domains are hydrophobic and capable showing

the interactions which both relative to DNA absorption.13 This could stabilize the DNA

molecules in the solution which is a really crucial property for DNA detection. The layer

number distribution of the GO sample sometimes have influence the performance of the

sample, while there are seldom papers reported the detailed mechanism. There are three

main methods to product GO are Brodie, Staudenmaier and Hummers Methods.11 All those

methods are involved in introducing the way to oxidized the graphite via di↵erent oxides.

For example, in Brodie and Staudenmaier methods, they use a oxide mixture of potas-

sium permanganate and sulfuric acid.14 These ways of chemical exfoliation from graphite to

GO provide outstanding process to produce a stable homogeneous GO aqueous suspension

which could use to fabricate continuous high quality thin film. And also the large amount

of chemically active oxygen defects give the sample the possibility of chemical functionlaized

treatment to improve the biological application.

4

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So called reduced graphene oxide has the surface that oxygen functional groups on orig-

inal GO are partly removed by chemical or physical treatment. The main purpose of this

reduction process is to restore the p-conjugated structure and the electrical conductivity.

Even though the remaining functional group on the surface of rGO might lower the elec-

tronic properties via reducing the conductivity, the electronic performance of rGO-based

biological sensors are benefit from the enhanced interaction between the remaining func-

tional groups and the analyte.15Thus the promising electrical conductivity and the chemical

active defects make the rGO as a vary attractive material using for fabricate the electronic

DNA sensors.1,2 The general reducing conditions for producing rGO is including two types of

treatments, high temperature thermal treatment and chemical treatments with the reducing

chemicals.16

3 Material Properties Related to DNA Detection

As we mentioned above, there are some properties of the graphene-based material have huge

influence on the performance of the graphene-based biological sensors. In this section, we

will summary how those properties related to the biological application.

3.1 Surface area

In figure 1, the fundamental surface structure of graphene and graphene oxide are shown. For

monolyer graphene, each carbon atoms are sp2-hybridized and are assigned on the surface by

layers. All those atoms are exposed outside. According to the BET test results, the specific

surface area of graphene could reach to 2630 m? which is vary high comparing to other

carbon -based material.11. This is at least one order of magnitude higher than other types

of the nanomaterials.17 As for graphene oxide, 600–900 m2 g1 of surface area was estimated.

This value of surface area roughly close to the theoretical value which is 890 m2 g1, even

though it is depended on the degree of oxidation of the GO and also on its aggregation

5

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level.18 The BET surface area measurements for reduced graphene oxides yielded 466 m2/g

which is related to the degree of GO exfoliation before the reduction. There is an potential

assumption being given to explain the decline of the surface area from graphene to GO and

rGO that the aggregation of the grahene oxide upon the reduction.19

3.2 Surface Chemistry

The surface chemistry situation for those graphene-based material are di↵erent even before

the functionalization due to the various surface structures and areas as we mentioned above.

Therefore, They demonstrated particular properties of surface-interaction among small gas

molecules to large biomolecules. There are two types interaction, covalent interation and

non-covalent interaction. In my work, I mainly focused on the non-covalent interaction

which is related to the DNA detection.

The surface of natural graphene is hydrophobic and the material has no significant sol-

ubility in most solvants.1,20 To get the possibility to fabricate the graphene-based devices

in soluble way, some scientist established some methods to fuctionalize the surface via non-

covalent interaction or covalent reaction to introduce some hydrophilic groups. The most

general method based on covalent reaction in terms of increasing the solubility involves the

oxidation of graphene to introduce the oxygenated species like carboxyl, epoxy and hydroxyl

groups on the surface of graphene producing graphene oxide.20 One drawback of this method

is the level of oxidation on surface can not be controlled.21 The non-covalent interactions

of graphene are formed based on van der Waals forces or – stacking.20,22 One of the benifit

of this type of interactions is that the surface structure of pristine graphene is preserved

not like the covalent interaction cases. From small gas molecules to large molecular weight

biomolecules, they all could be absorbed on the surface of graphene.23Here we main focus on

the DNA-graphene interaction. The nature of the interaction between the material surface

and DNA bases are not trully understood, it is still considered as non-covalent interaction.

Several types of forces are studied and regarded as the main contribution of the interaction

6

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including – stacking, electrostatic, van der Waals, and hydrophobic interactions.22 Among

all these forces, – stacking is contributed the most which explained why single-stranded DNA

shows stronger binding to graphene than double-stranded DNA where the intramolecular hy-

drogen bonding of bases are shown. In addition, The binding strength between the di↵erent

bases with graphene surface vary as the di↵erence of their polarizability.22

As product of graphene oxidation, graphene oxide shows better dispersability in variety

solvants due to the surfaces of GO contains hydrophilic regions, by which means that the

hydrogen bonding between surfaces and biomolecules or metal ions could form.1,24 Just like

we mentioned above, the surface of GO could be viewed as two types of domains, oxidized

regions that the carbon atoms are sp3 hybridized bonding with oxygen functional groups

and the unoxidized regions that the carbon there are sp2 hybridized.25 One of the most im-

portant reaction of GO is its reduction because the product reduced graphene oxide shows

higher electrical conductivity than GO. This reduction proecess could be accomplished via

chemical, electrocehmical or thermal reduction methods.21 And also due to high reactivity of

chemistry of GO, there are many ways to fucntionalize the surface though chemical reactions

which gives many possible modified structures utilizing for DNA detection and also other

molecules binding shown in figure 2. For example, Lu and his coworker reported that they

functionalized the GO surface with oligonucleotide molecular beacon (MB) which increas-

ing the seperation ability of ss DNA and dsDNA.26 This will talk more specifically later.

GO demonstrates the balance between the reasonable dispersability in solvants and relative

high non-covalent interaction domains, making this material as promising platform for high

sensitive and selective detection of DNA as I talked.27–29 The general mechanism of DNA

absorption on GO surface is roughly like for graphene, while the oxygenated species on the

GO’s surface contribute to the binding too.27,30 But still there are some works indicated

that they found the proof of DNA chemical grasping on GO.25 Hydrophobic forces and -

stacking play the most crucial roles in the interactions which can overcome the electrostatic

repulsion. After binding di↵terent types of DNA, the whole material demonstrates di↵er-

7

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ent electrochemistry, electronic conducting or optical properties which could be detected via

some specific measurments.1

2.jpg

Figure 2: Schematic illustration of GO-based electrodes for electrochemical applications.25

Copyright 2010 C. Da Chen.

3.3 Electrochemistry

One popular type of DNA sensor is known as electrochemical sensor which is fabricated based

on the detection of the change for electrochemistry properties after DNA absorption.1 There-

fore, to understand the electrochemistry properties of graphene-based material is important.

It is possible to investigate the electrochemistry properties based on the results from the

enormous amount of publishments on graphite and carbon based nanotubes. Because it was

shown that there is no di↵erence of electrochemistry properties from SWCNTs to graphite.31

Graphene shows a wide electrochemical potential of ca. 2.5 Vin 0.1 M PBS (pH 7.0), which

is comparable to the graphite.4 The charge-transfer resistance on graphene is much lower

that of graphite electrodes.32 As for GO and rGO, they both exhibit high electrochemical

capacitance with excellent cycle performance and rGO shows even higher electrochemical

8

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capacitance and cycling durability than carbon nanotubes (CNTs).The specific capacitance

was found to be 165 and 86 F/g for rGO and CNTs, respectively.25 Cyclic voltammetry of

graphene oxide sheets exhibits significant reduction waves starting at 0.60 V (vs. Ag/AgCl

reference electrode), reaching a maximum at 0.87 V.

4 Graphene-Based Sensors

With all these merits of graphene-based material, they have become crucial candidates for

DNA sensing application in liquid situation. As I mentioned above, the varietion of the

amounts of di↵erent DNAs on the material surface will change the electrochemical, electronic,

or optical behaviors which could be detected via some technologies to achieve the sensing

job. Here, we provide the review of the sensors in three categories based on the parameters

they detected, which are eletrochemical, electronic and optical DNA sensors. Furthermore,

many derivation graphene-based material obtained from the functionalization through the

formation of donor-acceptor complex ,these could improve the DNA sensor performance.33

The nanoparticles, organic compounds and biomolecules are utilized for functionalization.25

4.1 Electrochemical DNA Sensors

Graphene has a large electrochemical potential window of ca. 2.5 Vin 0.1 M PBS (pH 7.0)

in solution, therefore the detection of the molecules with either high oxidation or reduction

potentials becomes achievable.33 Graphene-based modified electrode is used for detection of

DNA hybridization for electrochemical changes. This type of DNA sensors o↵er really fast

response speed, high sensitivity and finest selectivity for the specific DNA hybridization.2,25

The main mechanism of novel electrochemical DNA sensor is that the DNA duplex (between

the probe and target DNAs) formed from the ss DNA grasping on the electrode surface, which

is labeled with an electrochemical indicator to recognize it. This immobilization of DNA onto

the surface caused an increase in the electrochemical impedance value, a further increase in

9

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electrochemical impedance value is observed after the hydridization.34 An interesting work

demonstrated by Wang’s group that a supersandnwich structure of biosensors which showed

high sensitivity, the detection limit is 100fM. The capture probe modified electrode was

denoted as cDNA/Au NPs/rGO/GCE where thiol is labeled at DNA.35

3.jpg

Figure 3: Schematic illustration of the experimental protocol. The platform can easily loadthe ssDNA probe and the detection mechanism is a↵ected by the ratio of polyaniline andgraphene (P/G). (above) ds-DNA releases from the platform when the mass ratio of P/Gis less than 1/20, (below) ds-DNA remains on the platform when the ratio of P/G is largerthan 1/10.36 Copyright 2015 C. Qing Zheng.

But there is a disadvantage of this type of label-needed sensors that the electrochemical

labels are involved making the architecture kind complex and the non-direct way to detect

target DNA decreasing the sensibility.35 Furthermore, the intrinsic electrochemical activity

of the nucleobases (primarily purine) at the GO-based electrodes provides the potential

application for the label-free electrochemical detection of nucleobases.2 This new type of

label-free DNA sensors has been proved to be one successful method to directly detect

the target DNA which requiring no labeling process or external indicators.35 Generally,

the multilayer composites architecture is used here. Peng and his coworker reported a high

10

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sensitive label-free electrochemical DNA sensor interface based on Au nanoparticles/toluidine

blue-GO composites thin film for the e↵ective detection of MDR1 gene. The peak current

was straightforward related to the concentration of the target DNA from 1.0 1011M to1.0

109M, with a detection limit of 2.95 1012M.35 Ping and his coworker fabricated dynamic

P/Gratio-based DNA sensor by deposition of polyaniline and pristine graphene nanosheet

(P/Gratios) composites in di↵erent mass ratios, DNA probe and bovine serum albumin

(BSA) layer by layer on the surface of a graphene-based electrode. It was capable to detect

C-DNA in a range from 0.01 pm to 1 m through changing ratios of polyaniline to graphene

and SNPs are also detectable for such sensor.36 The illustration of the detecting process is

shown in figure 3.

4.2 Electronic Sensors

The single layer graphene is a semi-metal with high carrier mobility (20,000cm2 V-1 s-1)and

carrier density 10-13cm-2 that are vary promising for fabrication of electronic sensors.33

Graphene electronic sensors are generally referred as Field e↵ect transistor(FET) which

mainly applied its field-e↵ect characteristics. The FET relies on an electric field to control

the shape and conductivity of a channel of one type of charge carrier could based on graphene-

based material. There are several principles could use to explain how this type FET working

as sensors. One general guess is that the graphene condcutance can be sensitively inflected by

minute gating signals. Some other claimed that the doping e↵ects, charge carrier scattering,

or change of local dielectric environment could also be used to realize the graphene-based

electronic detection.2 Mohanty and his coworker reported an electronic DNA sensor based

on a microsized graphene oxide sheet.The probe ss DNA was physical absorpted on the

GO sheet and the conductance of GO increased which means that the hybridization of

the target ssDNA as a result of doping e↵ect under dry condition.In addition, they also

demonstrated that the hybridization of a pair of target and probe ss DNA produced hole

doping e↵ect.3demonstrated another assumption on other electronic sensor that the detection

11

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(decrease of graphene conductance) is based on DNA induced n-doping on graphene instead

of the field-e↵ect and impurity screening mechanism.2

4.jpg

Figure 4: Schematic illustration of the graphene FET device operated by liquid gate37

Copyright 2016. Chun-Yu Chan.

Recent years, the operation of GFET sensors in aqueous condition has promoted for

biosensing. Zheng and his coworker developed a novel PNA-functionalized G-FET biosensor

based on CVD grown monolayer which could avoid the contaimination and large number of

defects on graphene surface. The author claimed that this device was used for the directional

technique and high-a�nity PNA-DNA hybridization for ultrasensitive, label-free, and highly

specific detection of DNA. It showed a great sequence-specific a�nity to target DNA and

achieved an excellent DNA detection sensitivity as high as 10 fM.38 Chan and his coworker

presented a graphene-based bio-field-e↵ect transistor (bioFET) for the detection of avian

influenza A virus subtype H7 gene based on CVD graphene and AuNPs. The scheme of

the bioFET device is shown in figure 4. The probes conjugated to the AuNPs were applied

to hybridize with the target gene in a sandwich assay format for signal amplification. This

biosensor demonstrated lower limit of detect of 64 fM, which is the lowest record for graphene-

based for DNA detection.37

12

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4.3 Optical DNA Biosensors

As I mentioned above graphene oxide exhibit outstanding optical properties. Unlike the zero-

gap graphene material, GO can fluoresce at a really wide range of wavelength, from near-

infrared to ultraviolet.33 This makes GO becoming a promising candidate as fluorescence

label for DNA detection. And also Go is capable of quenching fluorescence. The quenching

e�ciency of GO is preferable than the traditional organic quenchers. Therefore, GO can

play either an donor or accepter role in fluorescence energy transfer(FRET).33 Some claimed

that the ratio and type of its oxygenation of the surface could use to control the optical

characteristics of GO.39

5.jpg

Figure 5: Three possible mechanisms of hybridization between a probe DNA adsorbed by GOand its cDNA (target DNA). The oxygenated groups and defeats on GO are not drawn forclarity of the figures. In all the cases, the probe DNA with a fluorophore label is preadsorbedand the cDNA is added afterward. The tendency of GO adsorbing ds-DNA is lower thanthat of the adsorption of ss-DNA. (A) LangmuirHinshelwood mechanism. (B) EleyRidealmechanism. (C) Displacement mechanism.40 Copyright 2013. Biwu Liu.

An novel GO-based DNA biosensor was exhibited by Fei Liu and his coworker. The

GO sheets were applied in an array format to recognize the specific target DNA-DNA hy-

13

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bridization interation.When the probe DNA bond to the surface of GO is hybridized with

the AuNPs labeled complementary DNA strand, then the fluorescence emission intensity of

the GO is decreased remarkably.41 In this case the GO served as the energy donors and the

AuNPs played the role in FET as the energy acceptor. There are still some cases that the

GO is treated as quenchers. One example is from Wang that the planar GO surface of the

sensor allows simultaneous quanching of multiple ssDNA probes labeled with di↵erent dyes

leading a multicolor sensor for the detection of multiple target DNA.42 Lu and his coworker

demonstrated similar principle works with dye-labeled DNA.20 A really interesting work did

by Liu and his coworker was to determine a possible mechanisms of how DNA hybridization

took place in the presence of GO via a fluorophore-labeled DNA probe with experimental

proof and exhibited some other mechanisms. If the interaction based on the LangmuirHin-

shelwood mechanism, the cDNA is also adsorbed followed by di↵usion through the pathway

on GO. When the cDNA achieves a probe DNA, a duplex is formed on the GO surface and

then desorbed like in figure 5a. Another possible mechanism is the leyRideal mechanism,

where the adsorbed probe DNA directly reacts with the target cDNA that is dissolved in the

solution phase at the GO/water interface like in figure 5b. And another mechanism shown in

figure 5c is also impact on the whole process, that some of the probe DNA could be displaced

by the target cDNA into the solution phase to hybridize with the free cDNA in solution. For

DNA detection, Huang and his coworker exhibited a GO based optical biosensor for DNA

single-base mismatch study. By applying a 40-mer probe DNA (P1), both short (20-mer)

and long (60-mer) targets led to much lower fluorescence signals than the complementary

target T1 that was of the same length as P1. But still the exact mismatch location of the

target DNA still cannot be determined via this way.43,44

14

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5 Conclusions and Outlook

Graphene series material become really promising candidates for a wide range of applica-

tion from physic, chemistry to biography in a short history. As I reviewed above, the DNA

sensors fabricating using graphene-based material especially graphene oxide exhibite really

outstanding performence with many merits like high sensitivity and selectivity, wide de-

tection range, quick responsibility and low cost. Each type of the DNA detection sensor

demonstrates outstanding behavior to recognize varieties types of DNA strand with relatice

low concentration requirement.

But the exact mechanism of the each type of DNA sensor is poorly understood and

critical experimental proof are lack. For the future development, how to solve the problems

about graphene material production with highly controllable and scalable and to construct

more e�cient and direct detection structure of devices become more crucial. In witness the

development of the graphene-based DNA sensors, we could predict that they will turn into

a important series of DNA sensors in industry field someday in sooner future.

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