A robust covalent coupling scheme for the development of FRET

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A robust covalent coupling scheme for the development of FRET aptasensor based on amino-silane modified graphene oxide Leyla Nesrin Kahyaoglu, and Jenna L. Rickus Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02663 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 7, 2018

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A robust covalent coupling scheme for the development of FRET aptasensor based on aminosilane modified graphene oxide Leyla Nesrin Kahyaoglu†, ‡, Jenna L. Rickus*, †Agricultural

†, §, ‡

& Biological Engineering; §Weldon School of Biomedical Engineering;

‡Birck-Bindley

Physiological Sensing Facility,

Purdue University, West Lafayette, Indiana, USA.

KEYWORDS:

Graphene

oxide,

aptamer,

amino-silane,

glutaraldehyde,

EDC/NHS, covalent conjugation, physisorption, fluorescent aptasensor,

ACS Paragon Plus Environment

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ABSTRACT: In recent years, numerous aptamers have been physisorbed on graphene oxide (GO) to develop FRET based aptasensors based on the high fluorescence quenching efficiency of GO. However, physisorbed aptasensors show poor signal reversibility and reproducibility as well as nonspecific probe displacement and thereby, are not suitable for many analytical applications. To overcome these problems when working with complex biological samples, we developed a facile and robust covalent surface functionalization technique for GO-based fluorescent aptasensors using a well studied adenosine triphosphate (ATP) binding aptamer (ABA).

In the scheme, GO is first modified with amino-

silane, and further with glutaraldehyde to create available carbonyl groups for the covalent attachment of a fluorophore and an amino dual modified ABA. The surface

modification

method

was

characterized

by

zeta

potential,

X-ray

photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR).

The linearity, sensitivity, selectivity and reversibility of the resulting GO

based covalent aptasensor was determined and systematically compared with the physisorbed aptasensor. While both sensors showed similar performance in


2

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terms of sensitivity and linearity, better selectivity and higher resistance to nonspecific probe displacement was achieved with the developed covalent ABA sensor. The surface modification technique developed here is independent from the aptamer sequence and therefore could be used universally for different analytical applications simply by changing the aptamer sequence for the target biomolecule.

1. INTRODUCTION In recent years, novel carbon based nanomaterials (CBNs) have been extensively used as substrates for biosensors. Among several CBNs, graphene oxide (GO) has received enormous attention in sensor applications because of its remarkable optical and electronic properties 1. In a fluorescence resonance energy transfer (FRET) sensor scheme, GO acts as an acceptor for efficient quenching of nearby fluorescent species owing to the sp2 carbon domains2,3. Additionally, the π-electron rich surface of graphene oxide adsorbs a wide range of biomolecules through multiple noncovalent interactions, including π-π stacking, electrostatic interactions, and hydrogen bonding


3

1.

These properties

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make GO an excellent platform for many optical sensing applications. In particular, because GO is an efficient quencher of fluorescently labeled peptides and aptamers, a wide range of fluorescent aptasensors have been developed for the detection of proteins, metal ions, small molecules and nucleic acids

4-7.

Aptamers are single-stranded, short RNA or DNA oligonucleotides with superior features including high binding affinity and specificity arising from their unique three-dimensional conformations. As aptamers being quite small usually 20–80 nucleotides with 6–30 kDa molecular weights compared to antibodies or proteins, they offer several advantages on the detection of biological molecules including a wide range of selection, easy and cheap production and good biocompatibility

8.

In a typical GO based detection scheme, the sensing

mechanism depends on the quenching of fluorescently labeled recognition probes as a result of the adsorption onto GO surface. When exposed to analyte of interest, fluorescently labeled probes are released from GO surface leading

to

fluorescence

recovery.

While

this

physisorption-based

sensing

scheme has been demonstrated to be simple and effective in several studies,


4

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the adsorbed recognition probes cannot produce reversible signals. Additionally, the physisorption-based systems are subject to non-specific probe displacement and thereby, leading to false positive signal

9,10.

For these reasons, physisorbed

aptasensors are not well suited for sensing in complex biological samples or dynamic physiological sensing. One approach to compensate for the false positive signal produced by nonspecific probe displacement is to use reference DNA, labeled with a different fluorophore that shows no response to target molecule. This approach has been reported for the detection of nucleoside triphosphates successfully 11,12.

Even

normalization

though of

the

false

internal

positive

reference

signal,

helps

release

and

the

quantification

thereby,

loss

of

recognition probes by nonspecific probe displacement cannot be avoided

and the 13.

Thus sensors fabricated with this method might not provide good signal reversibility and reproducibility. GO, being the oxidized form of graphite carries a significant amount of oxygen containing groups on the surface, which enables better aqueous


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dispersibility as well as facile probe anchoring via covalent interactions

3.

Therefore, a better approach to produce reversible sensors is to covalently attach molecular probes onto the GO surface

10,13-17.

To date, native carboxyl

groups on the edge of GO have been used to covalently immobilize aminolabeled aptamers on GO surface via carbodiimide chemistry. However, this technique also requires the excessive washing steps including the use of complementary DNA (cDNA) with urea in the presence of heat to remove physisorbed probes on the remaining surface area of GO

10,13,18.

Here we

propose a new method based on surface modification of GO. We graft the GO first with amino-silane to block the surface and minimize the nonspecific probe displacement. Later, amine groups on the GO are treated with aldehyde groups to create available carbonyl groups on the GO surface for the covalent immobilization of aptamer (Figure 1a). In this new approach, we use the wellcharacterized adenosine triphosphate (ATP) binding aptamer (ABA) labeled with FAM (carboxyfluorescein) as the recognition probe and silane modified GO as the fluorescent quencher.

As the probe DNA remains on the GO surface upon


6

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target binding, the proposed covalent sensor can be regenerated. To verify our method, we monitor each step of the GO surface functionalization through a series of surface characterization experiments. Additionally, the performance of developed sensor is compared to that of a physisorbed sensor and the covalent aptasensor is optimized for the best performance. 2. EXPERIMENTAL PROCEDURES 2.1. Materials All aptamers were purchased from Integrated DNA Technologies, Inc. (IDT, Coralville, IA).

ATP binding aptamer (ABA) sequences were used as 5’-FAM-

ACC TGG GGG AGT ATT GCG GAG GAA GGT-NH2-3’ and with the oligothymidine T10 spacer (T10: 10 thymine) as 5’-FAM-ACC TGG GGG AGT ATT GCG GAG GAA GGT T10-NH2-3’. Graphene oxide (GO) was obtained from ACS Material (Medford, MA).

Glutaraldehyde (MP Biomedicals, Irvine, CA),

adenosine 5’-triphosphate disodium salt hydrate, dimethyl sulfoxide (Alfa Aesar, 99.9 %, Ward Hill, MA) and sodium cyanoborohydride (Acros Organics, 95%, Morris Plains, NJ) were purchased from Fisher Scientific (Pittsburg, PA).


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Bovine serum albumin was from Sigma-Aldrich (St. Louis, MO). Uridine-5'triphosphate trisodium salt dehydrate (UTP), cytidine-5'-triphosphate disodium salt (CTP) and N-(2-Aminoethyl)-3-(trimethoxysilyl)propylamine were obtained from Chem-Impex Int., Inc (Wood Dale, IL).

2.2. Aptamer binding affinity characterization by biolayer interferometry Biolayer interferometry (BLI) experiments were performed using Octet RED 384 instrument (ForteBio, Menlo Park, CA). ATP-binding aptamer was modified with biotin at 3’-end allowing immobilization on to SAX (High Precision Streptavidin Biosensor) sensor surface. Biotinylated aptamer was diluted in HKM buffer (25 mM Hepes, 100 mM NaCl, 5 mM MgCl2 at pH 7.4). All BLI experiments were performed in HKMBT buffer (25 mM Hepes, 100 mM NaCl, 5 mM MgCl2, 1 mg/ml BSA and 0.05% Tween 20 at pH 7.4). Various ATP concentrations ranging from 1 to 100 mM were prepared in HKMBT buffer. Samples were agitated at 1000 rpm during the experiment at 300C. SAX sensors were loaded with 50 nM biotinylated ATP-binding aptamer for 60


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seconds. Association and disassociation were both monitored 300 seconds. The ForteBio Data Analysis Octet Software was used to determine the binding affinity, Kd.

2.3.

Preparation of amino silane modified graphene oxide

6 mg of graphene oxide was dispersed in 100 ml of DMSO in an ultrasonic water bath (100 W output power, Branson 2510, Danbury, CT) for 30 minutes. DMSO was selected as solvent as it has been shown to enlarge the interlayer space of GO and thereby disperse GO well

19.

350 µl of N-(2-Aminoethyl)-3-

(trimethoxysilyl)propylamine was added to the well dispersed GO solution. The solution was refluxed at 120

oC

overnight under nitrogen gas. Amino-silane

modified GO was washed several times with DMSO, ethanol and DI water by ultracentrifugation to remove unreacted materials. Amino-modified graphene oxide was stored at room temperature in DI water (resistivity ≈ 18.2 MΩ/cm, Barnstead Micropure

water

purification

system,

Waltham, MA).


9

Thermo-Fisher

Scientific,

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2.4. Preparation of aptamer-modified graphene oxide The surface amino groups on the graphene oxide were reacted with glutaraldehyde to create reactive carbonyl functional group, C=O on graphene oxide surface. The resulting carbonyl groups reacted with amino-modified ATP binding aptamer on graphene surface and formed stable amine bonds by the reduction of imide bonds with sodium cyanoborohydride. The surface coverage of graphene oxide with amino-silane was approximated to be 10% from Table 1 in Section 3.1.4. A 10-fold molar excess of glutaraldehyde was added to ensure each amino group on GO reacts with a carbonyl group of glutaraldehyde. Briefly, amino-modified graphene oxide (250 µg/ml, 1.0 ml) was mixed with glutaraldehyde (50% in water, 0.5 ml) and incubated for 2 hours. The resulting graphene oxide mixture was washed 3 times with DI water. After final wash, carbonyl modified graphene oxide was resuspended in HKM buffer (1 ml) and amino-modified ATP binding aptamer was added (30 µl, 100 µM) and stirred for 2 hours. Following washing steps were applied; wash 1 in buffer, wash 2 in 1


10

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M ethanolamine (ETA) to block unreacted aldehyde, pH 8.4, wash 3 in 5 mM Tris-HCl, pH 9.4 (TRIS) with freshly made 20 mM sodium borohydrate to reduce the imide bond to stable amine bonds, wash 4 in HKM buffer. Flowthrough, first supernatant after GO-aptamer incubation, was collected before wash 1 for further analysis. Coupling

efficiency

was

Final sample was stored in HKM buffer at 40C.

estimated

by

measuring

optical

absorbance

of

supernatant after each washing step at 260 nm and converting obtained values to aptamer concentrations using Beer-Lambert law after background subtraction. Physisorbed aptasensors were prepared by mixing aptamer (0.5 µM) and graphene oxide (200 µg) in HKM buffer for 30 minutes at room temperature. Then the sensors were washed with HKM buffer for three times and stored at 4 °C until use.

2.5. Aptasensor characterization Fluorescence

emission

intensity

of

aptasensors

was

measured

using

a

microplate reader (Biotek Synergy Neo, λex/em = 485/523 nm). All aptasensor


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samples were incubated with various ATP concentrations for 30 minutes before fluorescence measurements. Specificity of the aptasensors was determined by comparing sensor response when exposed to same concentrations of CTP, UTP and ATP (2 mM). Nonspecific probe displacement of aptasensors was tested by adding different concentrations of BSA (1 mg/ml, 2 mg/ml) and incubating for 30 min before fluorescence measurement.

2.6. Surface characterization of graphene oxide The surface charge of graphene oxide after each chemical modification was measured using a Malvern Zetasizer (Nano Z, Malvern, UK). Zeta potential was determined by fitting to the Smoluchowski model and taking the data from the Malvern software without further correction. All zeta potential measurements were performed in DI water (pH 6.9) to minimize any possible pH effect.

To

further verify the surface modifications, we also performed X-ray photoelectron spectroscopy (XPS) (Kratos Axis Ultra DLD spectrometer, Kratos Co. Ltd., Manchester, UK) to monitor the chemical composition on the surface of


12

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graphene oxide. The C−C component of the C1s peak was set to a binding energy of 284.8 eV to correct for charge on each sample prior to data analysis. Fourier transform infrared (FTIR) (Thermo Scientific Nicolet 6700, Waltham, MA) spectral measurements were performed in transmission mode over the range from 4000 to 500 cm-1 with a resolution of 4 cm-1 with 50 repetitive scans.

2.7. Statistical Analysis All experiments were performed in triplicate and data were indicated as mean ± standard deviation (SD). All statistical analyses were carried out using statistical analysis software OriginPro (OriginLab Corporation, Northampton, MA). Analysis of variance (ANOVA) with Tukey’s post hoc test was used for statistical evaluation of experimental data (p < 0.05).

3. RESULTS AND DISCUSSION


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GO-based fluorescent aptasensors mainly rely on the adsorption and release of fluorophore labeled DNA probes from GO surface upon target molecule binding.

In this sense, most sensors are fabricated simply by the adsorption of

aptamers through noncovalent π–π stacking to the GO surface

1.

These

physisorbed sensors rely on desorption of aptamers from the GO surface when the aptamer binds the target molecules, resulting in decreased quenching of the fluorescently

labeled

aptamer

and

therefore

an

increase

in

measured

fluorescence. However, these physisorption-based aptasensors are susceptible to

nonspecific

probe

displacement

with

poor

signal

reversibility

and

reproducibility, which makes them less likely to be used for practical sensor applications

such

as

with

complex

biological

samples

or

continuous

monitoring18. Thus, a need for the development of aptasensors through covalent modification of GO surface has emerged.

Covalent surface modification of GO

can be achieved using the available epoxy and hydroxyl groups on its basal plane or its edge carbonyl groups. All these functional groups can be targeted and be easily dehydrated with silane coupling agents


14

20.

To date, silylation of

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GO

with

organosilanes

was

mainly

studied

for

polymer

composite

reinforcements to improve interfacial adhesion of GO with matrices21-23. Here, we propose to graft the GO surface with dual functional amino-silane, which will serve as both surface blocking and coupling agent in a sensing scheme (Figure 1a).

Active amine groups of the organosilane later will be transformed

to aldehyde and be coupled to amino-modified aptamer via Schiff base linkage. We use a well-characterized ATP binding aptamer (ABA) to develop, test and characterize the approach.

In our sensing scheme, binding of ATP induces a

conformational change in the ABA, which alters the relative proximity of the fluorophore from the GO surface leading to a fluorescence response (Figure 1b).

Because the ABA is tethered to the GO, the ABA can re-associate with

the GO after ATP is removed, thus allowing for dynamic and reversible sensing.

a.


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b.


16

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Figure 1. Schematics of functionalization (a) and sensing principle (b) of covalent ABA aptasensor a. GO surface is first grafted with amino-silane and then modified with glutaraldehyde to create a linker chain with exposed carbonyl groups for the covalent attachment of a fluorophore and amino dual labeled adenosine triphosphate (ATP) binding aptamer (ABA) through amide bond formation. b. Upon ATP binding, FAM label of ABA moves away from GO surface, resulting in decreased fluorophore quenching by the GO and increased measured fluorescence emission.

3.1. Surface characterization of GO 3.1.1. Zeta potential measurements monitor surface charge changes and the introduction of surface groups with each functionalization step.


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The surface charge modulation in each functionalization step was determined using zeta (ζ) potential measurements (Figure 2a). The GO surface was first grafted

with

an

amino-silane,

N-(2-Aminoethyl)-3-(trimethoxysilyl)propylamine,

covalently through a silylation reaction

24,25.

Silylation reaction was performed by

replacing the labile hydrogen in the hydroxyl and carboxyl groups on GO surface with the trimethoxysilyl groups of amino silane (Figure 1a)

22,25.

The silane layer was used to minimize nonspecific biomolecule adsorption to GO, provide some length to the linker chain, and to introduce available amine binding sites for reaction with glutaraldehyde in the next step.

Before

aminosilane modification, GO showed negative surface charge of -36.23±4.55 mV owing to the native surface carboxylic acid groups. The surface charge increased significantly as expected to 9.82±0.72 mV after grafting the graphene oxide surface with positively charged amine groups of aminosilane.

Next the

GO-silane-amine was reacted glutaraldehyde and the surface charged dropped significantly to 1.77±0.42 mV, consistent with the introduction of the carbonyl groups.

Finally, we performed covalent attachment of the amino modified ATP


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binding aptamer (ABA) through the reaction of aldehydes with primary amines. After this reaction the surface charge slightly increased to 1.91±0.54 mV. each

step

the

surface

charge

changes

showed

an

expected

After

outcome

suggesting successful anchoring of each chemical group to the GO surface. 3.1.2. GO is reduced after amino-silane treatment with heat. After silane modification, the color of the GO sample turned from brownish yellow to black (Figure 2), which would be an indication of the reduction of GO 26.

To test the reduction of GO, ultraviolet-visible (UV-Vis) spectroscopic

measurements were performed before and after amino-silane treatment with heat. GO showed the characteristic absorption peak at 230 nm corresponding to the π- π* transitions of C=C aromatic rings and a shoulder band around 300 nm originating from n- π transitions of C=O bonds before treatment

27(Figure

2b). After treatment with heat and amino-silane in DMSO overnight, the absorption peak of aromatic rings was red-shifted to 260 nm and the band at 300 nm disappeared as a result of the reduction of GO due to the removal of


19

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oxygen containing groups

27,28.

The reduction of GO would be attributed to

DMSO, which might serve as a reducing solvent in the presence of heat a.

29.

b.

Absorbance (a.u.)

1.0 0.8 0.6 0.4 0.2 0.0

GO amino-silane modified GO 200

300

400

500

600

700

800

Wavelength (nm)

-CH2

C-O-C

C-O-C

-OH Si-OH

CH -OH C-O-C

d.

C=O C=C

c.

1.5

GO

1.0

CD (mdeg)

CN Si-O-C Si-O-Si Si-OH

CH

GO-Glut

CO-NH CO-NH

NH

GO-silane

CH

GO-Apt

3500

3000

ABA ABA on GO ABA with ATP ABA on GO with ATP

0.5

0.0 200

220

240

260

2500 2000 1500 Wavenumber (cm-1)

PO2

-0.5 PO2

Transmission (%)

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 20 of 73

1000

-1.0

500


20

Wavelength (nm)

280

300

320

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Figure 2. Characterization of GO surface modification and ABA. a. Surface charge

modulations

on

GO

surface

were

monitored

after

each

step

of

functionalization through zeta potential measurements suggesting successful surface

grafting

with

functional

groups.

Error

bars

indicate

the

standard

deviation of the mean (SD). Treatments (n = 3) with no letters in common are significantly different within the given group (p < 0.05).

b. UV-Vis spectra of

the graphene oxide (GO) shows a maximum absorption peak at 230 nm and a shoulder peak around 300 nm corresponding to the π- π* transitions of C=C aromatic rings and n- π transitions of C=O bonds, respectively. After aminosilane treatment with heat, the absorption peak is shifted to 260 nm with the disappearance of shoulder peak suggesting the reduction of GO.

Inset shows

the corresponding photographs of GO with brownish yellow color and reduced GO with black color. c. FTIR spectra for GO, GO-silane (amino-silane GO), GO-Glut (glutaraldehyde modified GO), GO-Apt (aptamer modified GO) suggest successful modification of GO with ABA. d. Circular dichroism spectra of free ABA and ABA modified GO suggests a parallel G-quadruplex structure before


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addition of ATP. The mixed form of parallel and antiparallel G-quadruplex is observed after the addition of 2 mM ATP.

3.1.3. FTIR was used to identify the chemical bond variations on GO surface at each functionalization step and suggests successful surface modification with ABA. To identify the chemical bonds on GO surface after each functionalization step, Fourier transform infrared spectroscopy (FTIR) was performed (Figure 2c). GO contains hydroxyl and ether functional groups on both sides of surface while carboxyls on the edge of the sheet (Figure 1a). The analysis of GO with FTIR showed these characteristic bands of stretching vibrations from hydroxyl groups at 3350 cm-1, carboxyl/carbonyl stretching vibration at 1730 cm-1, skeletal vibrations from unoxidized C=C/C-C sp2 carbon of graphene and bending modes of water molecules at 1620 cm-1 and hydrocarbon vibration from phenols, ethers, and epoxy groups at 1250 cm-1, 970 cm-1 and 890 cm-1, respectively

30,31.

Additional

bands

of

bending

vibrations

from

aliphatic

hydrocarbon groups at 1455 cm-1, hydroxyl vibration from the C-OH groups at


22

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1375 cm-1, and C-O stretching vibrations from hydroxyl groups at 1080 cm-1 were observed

32-34.

After amino-silane modification, bands of all oxygen

containing bonds were either diminished (hydroxyl vibrations at 3350 cm-1 and at 1375 cm-1) or vanished (carbonyl vibration at 1730 cm-1 and epoxy vibrations at 1250 cm-1, 970 cm-1 and 890 cm-1) confirming the reduction of GO as well as the successful grafting of amino-silane on GO via ring opening reaction of epoxide under the attack of the amine group at the elevated temperatures 24,31,35.

The new bands at 1650/1575 cm-1 were assigned to the amide bond

formation between carboxyl/carbonyl groups of GO and amine groups of silane as a result of heat treatment

31.

The disappearance of carbonyl vibration at

1730 cm-1 was also an evidence of heat-induced amidation of carbonyl groups. Thus, amine groups of silane can modify the GO surface via nucleophilic substitution of the epoxy groups and heat induced amidation of carboxyl groups31. An additional broad band appeared at 3650 cm-1 corresponding to hydroxyl group of surface silanols

36,

which suggests incomplete condensation

reaction with Si-OH stretching peak at 920 cm-1 as well as the asymmetric


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stretching vibrations of Si-O-Si at 1040 cm-1

37.

Further, the band at 3300 cm-1

was assigned to N-H stretching of secondary amine

38

and the band at 1195

cm-1 to C-N stretch of secondary amines and at 1110 cm-1 to asymmetric stretching vibration of Si-O-C as a direct evidence for the successful silanization of GO

39.

Next amino-silane modified graphene was reacted with glutaraldehyde to create carbonyl groups on the surface. After aldehyde modification, the peak of carboxyl stretching vibration at 1720 cm-1 appeared again confirming the successful carboxylation of GO. All epoxy bands came back with an additional one around 3060 cm-1 assigned

to

C-H

30.

The shoulder peak at around 2725 cm-1 can be

stretching

of

formyl

groups

suggesting

the

successful

aldehyde-functionalization on GO surface40. Additionally, increased band intensity around 1110 cm-1 would be the result of anhydride bond formation. At the last step, amino-modified ABA was coupled to carboxylated GO surface through a Schiff base reaction. The stretching vibration of phosphate groups from DNA backbone appeared at 1210 cm-1 and 1056 cm-1 (PO2)


24

41,42.

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Overall, FTIR spectra showed the successful grafting of GO with ABA via series of chemical modifications. 3.1.4. XPS confirms the surface grafting of GO with ABA. Further evidence of successful surface grafting was demonstrated by X-ray photoelectron spectroscopy (XPS). The characteristic C1s peaks of GO were observed at 284.8, 286.3 and 288.2 eV corresponding to C-C (graphite carbon), C-O (epoxy/ether group) and C=O (carbonyl group) bonds, respectively (Figure S1a). In the C1s spectrum of amino-modified GO (Figure S1c), four types of carbon were fitted with different chemical states at around 284.8 eV (C–C), 285.5 eV (C–N), 286.6 eV (C–O) and 288.8 eV (C-N-O/C=O). Similarly, the O1s spectrum of GO before amino-silane modification showed only the peaks at around 532 eV, corresponding to O in O-C-O (532.5 eV), C=O (531.4 eV) and OH (535.9 eV) bonds (Figure S1b) whereas after the silane modification, the various components of O1s appeared at 532.1 eV for Si-O-C, 532.7 eV for SiO-Si/C-OH/C-O-C/O=C-N, 534.5 eV for O=C-OH and 536.2 eV for hydroxyl groups (Figure S1d)

35,43.


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Page 26 of 73

Compared to GO, new N1s (at 399.9, 402.4 and 405.7 eV corresponding to amine and amide groups of –NH-/NH2, N(O)=C and NO2, respectively)(Figure S2a) and Si2p (102.8 and 105.7 eV, correspond to Si-O-C and Si-O-Si/-OH) peaks were observed (Figure S2b)

43-46.

The presence of Si-O-C peak suggests

the successful surface modification while the observed peak for Si-O-Si/-OH indicates the hydrolysis of silane on GO surface. Additionally, the peaks of amide groups on N1s spectra also confirms the amino-silane of GO through nucleophilic substitution of the epoxy groups and heat induced amidation of carboxyl groups as discussed in Section 3.1.3. The resulting amino-silane bearing GO surface shows also a significant reduction in the peak intensity of oxygen groups as a result of the reaction between amines of silane and epoxy groups on GO (Table 1)

21,45.

Table 1. Atomic percentages of C, O, N, Si and P for GO, GO-silane (aminosilane GO), GO-Glut (glutaraldehyde modified GO), GO-Apt (aptamer modified GO).

Sample

C1s (%)

01s

N1s


26

Si2p

P2p

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(%)

(%)

(%)

(%)

58.9

41.1

-

-

-

modified 53.6

30.3

7.9

8.2

-

Glutaraldehyde modified 48.0

35.4

8.4

8.2

-

25.9

11.4

2.1

0.6

GO Amino-silane GO

GO ABA modified GO

60.0

In the next step, primary and/or secondary amines coming from silane reacted with glutaraldehyde on GO to create carboxyl groups. The C1s spectra of aldehyde modified GO surface showed three carbon components, one carbonyl group C=O contribution at 288.0 eV, and the unchanged contributions from C-C at 284.8 eV, and C-N at 285.6 eV, respectively (Figure S1e). The additional peak at 530.6 eV on O1s spectra showed the formation of carboxylate species, COO- after glutaraldehyde treatment on amino-GO surface (Figure S1f)47. High resolution elemental scan of the N1s also showed the additional peaks at 398.5 eV, possibly as a result of imide bond formation, –C=N and at 401.3 eV for the protonated amines as the byproduct of carboxylation reaction of amines (Figure S2c)

43,48.

Further, as amine was converted to carboxylic functional group, a


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characteristic O=C-O/O=C-N peak of carboxylic moieties appeared at 288.0 eV, together with visible C-N binding energy at 285.0 eV in the high resolution C1s narrow scan (Figure S1e). After addition of the amino modified ABA, new P2p spectra was emerged with doublet peaks at 134.0 eV for P 2p3/2 and at 134.7 eV for P 2p1/2 arising from the phosphate backbone of immobilized ABA on GO surface (Figure S2g)

49,50.

Overall, the surface characterization of GO through

XPS and FTIR techniques are in good agreement with each other and support the successful covalent grafting of GO with ABA. 3.2. Aptamer characterization 3.2.1. Fluorophore labeling does not change the binding affinity of ABA. The labeling of analyte-sensitive aptamer with a fluorophore is required in our proposed sensing scheme. GO acts as a photoluminescence acceptor in the visible range.

To build an energy transfer based sensing mechanism, we used

6-carboxylfluorescein (6-FAM) for labeling the ATP binding aptamer, which has the excitation and emission maximum (485/523 nm) within the visible light range. However, the fluorophore labeling could affect the selective target


28

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Langmuir

recognition and binding abilities of aptamer by disturbing the folding of the DNA. In order to examine how 6-FAM labeling impacts the ATP-aptamer interaction, we use the biolayer interferometry (BLI) technique. The binding affinity (Kd) of 6-FAM labeled ABA was measured to be 58.7±12.5 µM (R2=0.93) (Table S1). Recently, the Kd of ABA without label has been reported to be 55.2±4.2 µM using surface plasmon resonance51. This finding from the literature was consistent with the measured affinity based on BLI suggesting that fluorescent labeling does not change the ABA binding affinity drastically. 3.2.2. The 3D structural conformation of ABA that is necessary for ATP recognition is preserved after covalent conjugation to GO surface. The specific conformation of DNA sequence defines the aptamer selectivity and sensitivity and thereby, disruptions to structure can drastically affect the aptamer binding characteristics. In our sensing scheme, these disruptions might be caused by the covalent immobilization of aptamer to the modified GO surface via glutaraldehyde. As a homocrosslinker of two primary amine groups, glutaraldehyde might not only crosslink one amine group from organosilane and


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Page 30 of 73

the other amine group from 3’end of labeled aptamer, but also might react with the primary amines in guanine, cytosine and adenine nucleotides of aptamer. Thus, we examined the effect of covalent immobilization on 3D structural conformation of aptamer. The ABA forms a G-quadruplex motif, which is crucial for the recognition of the target biomolecule, ATP selectively

52,53.

We used

circular dichroism spectroscopy (CD) to monitor and compare the secondary structure of ABA aptamer as free strands in the solution and as covalently bound to GO surface (Figure 2d). Both spectra showed two positive peaks near 270 and 210 nm and a negative peak around 240 nm suggesting a parallel Gquadruplex structure when there was no ligand54. Retained conformational structure of ABA after immobilization to GO surface would be an indicator that there might not be any available primary amines in guanine, cytosine and adenine nucleotides of aptamer during aldehyde coupling. Because primary amines might participate in hydrogen bonding for base pairing (guanine-cytosine and adenine-thymine)

55,56

in 3D conformation of aptamer and four consecutive

guanines in ABA sequence fold into G-quadruplex structure through hydrogen


30

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Langmuir

binding between primary and secondary amines of guanines

57,58.

The addition

of ATP induced the structural translation from parallel to a mixed form of parallel and antiparallel G-quadruplex with a positive maximum around 280 nm and a negative maximum at 250 nm59. Therefore, CD spectra suggest that single-stranded ABA retained parallel G-quadruplex structure and ATP induced antiparallel G-quadruplex motif after covalent immobilization on GO surface. Additionally, the CD results show that the covalent immobilization method developed in this study can be feasible for aptamers that require G-quadruplex formation to achieve specific target recognition. 3.3.

Characterization of GO and aptamer complex

3.3.1. Silane modification acts as a surface-blocking agent and improves the sensor robustness by minimizing the adsorption of probes to GO surface. The

coupling efficiency of ABA

to GO

surface

is

crucial to

produce

aptasensors reproducibly. We define the efficiency as the ratio of ABA coupled to the total amount added. After functionalization with fluorophore labeled ATP binding aptamer, GO was treated to remove non-covalently bound aptamer.


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Page 32 of 73

The fluorescence emission signal of the resulting supernatant was measured after each washing step to estimate the coupling efficiency. Commonly, the complementary DNA sequence (cDNA) with heat and urea has been used to desorb physisorbed aptasensors from GO surfaces after covalent attachment of probe DNA aptamers

10,13.

We used this method here to test the coupling

efficiency of our covalent attachment method and compared the outcome directly to a physisorbed ABA-GO complex.

First the physisorbed and

covalent aptasensors were produced using 3 µM of ABA on 250 µg/ml of GO. Next, both aptasensors were incubated with cDNA and then exposed to urea (10 M) in the presence of heat (700C for 10 minutes) three times to remove the hybridized cDNA from GO surface. After each washing steps, supernatants were

collected.

The

fluorescent

emission

of

collected

supernatants

was

measured and fit to a background normalized fluorescent calibration curve of ABA in order to determine the ABA concentration (Figure S3). Prior to cDNA exposure, almost half of the added aptamer (around 45%) was collected at the first supernatant for covalent sensors while the fraction of the aptamer removed


32

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from GO was around 10% for physisorbed sensors at this step suggesting a large portion of the ABA was effectively adsorbed on GO surface (Figure 3a). On the other hand after cDNA treatment, ABA on GO surface was removed to great

extent

for

physisorbed

sensors

(≈65%)

whereas

the

removal

rate

remained around 10% for covalent aptasensors. After all washing steps, only around 2% of total ABA added remained physisorbed to GO surface while the coupling efficiency was determined to be 23% for covalent aptasensors on silane-modified GO surface (Figure 3a). The

covalent

functionalization

of

GO

with

biomolecules

performed through a well-known carbodiimide procedure

60-62.

is

commonly

This method relies

on the reaction between the carboxyl groups on the edges of GO and amino groups of biomolecules such as amino-modified DNA via EDC/NHS chemistry 10,13.

As the probe functionalization occurs only at the edge, the vast majority of

GO surface is still subject to physisorption of DNA and thereby, requires cDNA treatment to remove physisorbed probe DNA from GO surface

10,13.

However,

we grafted the surface of the GO with silane, with the goal of using the silane


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as

a

surface-blocking

functionalization

scheme.

agent To

before test

the

the

Page 34 of 73

addition

blocking

of

effect

aptamer of

silane

in

our

grafting,

physisorbed aptasensors were produced with unmodified and silane-modified GO. Additionally, the two physisorbed aptasensors were not treated with cDNA and the combination of urea with heat after coupling. Instead, the physisorbed aptasensors were washed with different buffers (HKM, ETA and Tris) to observe the probe adsorption phenomena better (Figure 3b). Only around 1% of initial amount of ABA added (3 µM) remained physisorbed on silane-modified GO surface after final wash whereas the remaining percentage of total ABA added was around 14% on GO without treatment. The significant reduction in the remaining physisorbed ABA implies that the silane modification minimizes effectively the noncovalent π–π stacking of aptamers to GO surface. We

also

performed

the

covalent

functionalization

with

different

ABA

concentrations to confirm the robustness of the method. When the covalent aptasensors were produced from the initial ABA concentrations of 1.5 and 3 µM using 250 µg/ml of surface modified GO, the coupling efficiency was around


34

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Langmuir

60% and 30%, respectively (Figure 3c). This result shows that almost the same amount of ABA, around 0.9 µM, was conjugated to GO surface regardless of initial DNA concentration (Figure 3c).

Additionally, compared to the washing

treatment combination of cDNA with urea and heat, the amount of ABA immobilized on GO surface covalently showed no significant difference when washing steps were performed only with buffers (Figure 3d). However, the coupling efficiency of ABA to GO surface increased significantly from 2% to 14% for physisorbed sensors when the washing treatment was switched from the harsh combination of cDNA with urea and heat to mild treatment of buffers only (Figure 3d). Overall, regardless of washing treatment and initial probe concentration added, the same amount of ABA can be coupled to silanemodified GO surface covalently and reproducibly with our method. a.

b.


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c.

d.

Figure 3. Characterization of GO and ABA complex. a. Coupling efficiency (%) after each washing step for physisorbed and covalent aptasensors. Around 23% of total ABA added was coupled to GO after all washing steps suggesting successful covalent immobilization of the probe. b. After the series of buffer washing, only around 1% of total ABA added initially remained on silane-


36

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Langmuir

modified GO suggesting that grafted amino-silane on GO serves a surface blocking. c. Regardless of total ABA added initially, the same amount is immobilized on silane modified GO. d. Coupling efficiency (%) after mild (buffers only) and harsh (cDNA with urea and heat) washing treatments for physisorbed and covalent aptasensors.

Coupling efficiency of physisorbed

aptasensors was significantly affected by washing treatments. FT: Flow-through after incubation of the probe and GO. HKM buffer: 25 mM Hepes, 100 mM NaCl, 5 mM MgCl2, pH 7.4. cDNA is the complementary DNA treatment. UH is the treatment with 10 M urea in the presence of heat (70oC for 10 minutes). ETA buffer: 1 M ethanolamine, pH 8.4. TRIS buffer: 5 mM Tris-HCl, pH 9.4. Error bars represent the standard deviation of the mean (SD). Treatments (n = 3) with no letters in common are significantly different within the given group (p < 0.05).

3.3.2. Covalent aptasensors are resistant to nonspecific probe displacement and can be regenerated simply by washing in buffer.


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Page 38 of 73

An ideal sensor should only respond to target molecules for practical applications. To test the sensor stability against non-target biomolecules, we measured and compared the response of the aptasensors (physisorbed vs covalent) to bovine serum albumin (BSA) as model cellular protein. Addition of BSA (2 mg/ml) caused a significant increase in the response of physisorbed aptasensor showing that physisorbed sensors are subject to nonspecific probe displacement. However, the covalent sensors showed only a small change in sensor response with BSA suggesting better stability and resistance to nontarget biomolecules (Figure 4a). The high resistance to nonspecific probe displacement would also be explained by silane modification, which occupies the GO surface and minimizes non-target molecule adsorption.

Furthermore,

signal reversibility is another crucial design parameter for certain biological applications such as real-time monitoring of analyte concentration. To monitor the reversibility, covalent aptasensors were regenerated briefly by washing in HKM three times.

Although sensor response to 10 mM ATP decreased after

the first cycle of buffer washing, the reduction in signal response compared to


38

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time zero was insignificant (p

A robust covalent coupling scheme for the development of FRET

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