Accepted Manuscript

Title: Single-Walled Carbon Nanotube based TransparentImmunosensor for Detection of a Prostate Cancer BiomarkerOsteopontin

Author: Abhinav Sharma Seongkyeol Hong Renu SinghJaesung Jang

PII: S0003-2670(15)00184-1DOI: http://dx.doi.org/doi:10.1016/j.aca.2015.02.010Reference: ACA 233724

To appear in: Analytica Chimica Acta

Received date: 25-10-2014Revised date: 29-12-2014Accepted date: 6-2-2015

Please cite this article as: Abhinav Sharma, Seongkyeol Hong, Renu Singh,Jaesung Jang, Single-Walled Carbon Nanotube based Transparent Immunosensor forDetection of a Prostate Cancer Biomarker Osteopontin, Analytica Chimica Actahttp://dx.doi.org/10.1016/j.aca.2015.02.010

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Single-Walled Carbon Nanotube based Transparent Immunosensor for

Detection of a Prostate Cancer Biomarker Osteopontin

Abhinav Sharma1, Seongkyeol Hong1, Renu Singh1, Jaesung Jang 1,2,3,†

1Department of Mechanical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Republic of Korea

2Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Republic of Korea

3School of Materials Science and Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Republic of Korea

† Correspondence should be addressed to jjang@unist.ac.kr. Tel: +82-52-217-2323 Fax: +82-52-217-2409

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Graphical abstract

Highlights

A transparent CNT immunosensor is presented for a prostate cancer biomarker osteopontin.

This immunosensor showed a highly linear and reproducible behavior from 1 pg mL-1 to 1 µg mL-1.

The limit of detection of the immunosensor was 0.3 pg mL-1. This immunosensor demonstrated high selectivity against bovine serum albumin

and human serum.

Abstract

Osteopontin (OPN) is involved in almost all steps of cancer development, and it is being

investigated as a potential biomarker for a diagnosis and prognosis of prostate cancer. Here,

we report a label-free, highly sensitive and transparent immunosensor based on single-

3

walled carbon nanotubes (SWCNTs) for detection of OPN. A high density of -COOH

functionalized SWCNTs was deposited between two gold/indium tin oxide electrodes on a

glass substrate by dielectrophoresis. Monoclonal antibodies specific to OPN were

covalently immobilized on the SWCNTs. Relative resistance change of the immunosensors

was measured as the concentration of OPN in phosphate buffer saline (PBS) and human

serum was varied from 1 pg mL-1 to 1 µg mL-1 for different channel lengths of 2, 5, and 10

μm, showing a highly linear and reproducible behavior (R2 > 97%). These immunosensors

were also specific to OPN against another test protein, bovine serum albumin, PBS and

human serum, showing that a limit of detection for OPN was 0.3 pg mL-1. This highly

sensitive and transparent immunosensor has a great potential as a simple point-of-care test

kit for various protein biomarkers.

Keywords: cancer; osteopontin; carbon nanotubes; dielectrophoresis; transparent sensor;

field effect transistor sensors.

1. Introduction

Cancer is one of the most life-threatening diseases in humans, and several types of

cancers are reported yet among which lung, liver, blood, and prostate cancers are most

common and have caused most of the deaths worldwide [1]. Prostate cancer is the second

most common cancer in USA after lung cancer [2]. Diagnosis of prostate cancer is a critical

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limiting factor for this disease because there is no sign of cancer progression for many years

in the initial stage. This often results in delayed diagnosis, and hence the cancer can spread

from the prostate to surrounding organs without being properly treated. There are several

techniques to diagnose prostate cancer including ultrasound, biopsy, and prostate specific

antigen (PSA) test. The PSA test is commonly used, and prostate cancer patients generally

have abnormally elevated PSA level in serum (>4 ng mL-1) [3]. However, it is well known

that PSA tests are prone to both false positives and false negatives [4].

Enzyme-linked immunosorbent assay (ELISA) is a commonly used method for

detection of a certain biomarker protein, but this assay has specific requirements such as

availability of pure samples, long processing time, special equipment and trained personnel

[5,6]. Therefore, it is highly desirable to make a simple, cost-effective, and highly sensitive

immunosensor that can detect a biomarker protein. Recently, carbon nanotube (CNT)- and

nanowire-based field effect transistor (FET) biosensors have shown excellent sensitivity

and selectivity with a low detection limit of PSA for prostate cancer [7-10]. A CNT-FET

based biosensor also showed a detection limit of 30 fM of osteopontin (OPN) for prostate

cancer using a genetically engineered single-chain variable fragment antibody [11].

OPN is a potential new biomarker of prostate cancer [12-14]. It is a secreted, 60-

kDa phosphoprotein that cancer cells use to facilitate their expansion and can be expressed

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in a variety of tissues such as bones, brain, kidney, lung and liver [15]. It is involved in

almost all the steps of cancer development, and it is being investigated both as a new

therapeutic target and as a potential biomarker for a diagnosis and prognosis of prostate

cancer [14-16]. The measurements of free OPN, which is not bound to complement factor

H, in plasma showed an increase with the stage of prostate cancer [13] although early

detection with OPN measurements has not been fully established, requiring more studies

[17]. It should also be noticed that OPN measurements are important for the prediction of

prostate patient survival and for detection of other cancers. Traditional protein detection

methods such as ELISA have shown to be problematic for its quantification [18] and there

have been very few studies on the cost-effective, easy to implement immunosensors for

OPN detection with comparable or better sensitivity than an ELISA assay.

In this study, we report a label-free, highly sensitive and transparent electrical

immunosensor to detect OPN using single-walled carbon nanotubes (SWCNTs). The

electrical detection based on CNTs has several advantages such as ease of fabrication, well-

understood carbon surface chemistry and simple measurements, which enables miniaturized

and inexpensive biosensors. The SWCNTs were deposited between two transparent source

and drain electrodes on a glass substrate by dielectrophoresis (DEP), making a channel for

OPN attachment and detection. Although chemical vapor deposition (CVD) is a most

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common technique to grow highly aligned SWCNTs [19] and offers good control on

positioning, it requires specialized materials and high temperature for the growth. On the

contrary, DEP allows the direct alignment of a high density of SWCNTs at room

temperature, and it is generally much simpler and more cost-effective. Monoclonal

antibodies specific to OPN were then covalently immobilized on the SWCNT surfaces by

EDC/NHS treatment. Relative resistance change of the immunosensors was measured for

different channel lengths of 2, 5, and 10 μm and constant channel width of 100 µm as the

concentration of OPN in human serum and phosphate buffer saline (PBS) was varied from

1 pg mL-1 to 1 μg mL-1. The selectivity of the immunosensors was also tested against

bovine serum albumin (BSA), human serum, and PBS.

2. Materials and Methods

2.1. Materials

SWCNTs (98% semiconducting, length: 5-30 µm, diameter: 1-2 nm, COOH

content: ~2.75 wt%) were purchased from M K Impex Corp., Canada. 1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and BSA were

purchased from Sigma Aldrich, USA. Normal human serum was purchased from Merck

Millipore, USA. Mouse monoclonal antibodies (AKm2A1) to OPN were purchased from

Santa Cruz Biotechnology, USA, and OPN was purchased from AbD-Serotec, USA.

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Dimethylformamide (DMF) (98%) and tween 20 were purchased from Biosesang, South

Korea. PBS (pH 7.4, 10x) was purchased from Life Technologies, South Korea. Deionized

water (dH2O) (resistance: ~18.2 MΩ) from the Millipore water purification system was

utilized for preparation of the desired aqueous solutions (molecular biology grade). All the

solutions and glassware were autoclaved prior to being used.

2.2. Fabrication of the SWCNT immunosensor

The immunosensors were fabricated using the conventional photolithography and

lift-off process of two gold/indium tin oxide (ITO) electrodes on a glass substrate. First, 500

µm thick glass wafers (Pyrex 7740, INEXUS, Inc., South Korea) were cleaned by a piranha

solution for 10 min, rinsed with dH2O, and dried with a nitrogen gas. The two electrode

patterns were generated by photolithography followed by electron beam evaporation of

gold (thickness: 2 nm) and RF-sputtering of ITO (thickness: 100 nm) on the glass wafer.

These two electrodes were 100 µm wide and separated by 2, 5, and 10 µm. A SWCNT

suspension was prepared by suspending the SWCNTs in DMF (10 µg mL-1) by 90 min

sonication in a water bath followed by centrifugation at 5,000 rpm for 1 h, and the

supernatant was discarded. After dropping 20 µl of the SWCNT suspension between these

two Au/ITO electrodes, a thin film of parallel-aligned SWCNT channels was formed by

applying 10 V (peak to peak value) at 200 kHz for less than 1 min.

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2.3. Bio-functionalization

After the deposition of SWCNTs, the immunosensors were rinsed with dH2O, dried

with a nitrogen gas stream, and treated with a mixed solution of 1 mL of EDC (5 mg mL-1)

and 1 mL of NHS (12 mg mL-1) in PBS buffer for 15 min at room temperature to activate

the carboxylic acid groups of SWCNTs followed by washing with PBS buffer (pH 7.4, 1x).

Monoclonal OPN antibodies (10 µg mL-1) in PBS were incubated on the CNT channels for

overnight at 4°C. After the antibody immobilization, the loosely attached antibodies were

removed by thoroughly rinsing with PBS buffer (pH 7.4, 1x) and air-dried at room

temperature. The attachment of the OPN antibodies occurred via the covalent binding of –

NH2 group with NHS succinimide ester on the SWCNTs. Tween 20 (0.5% in 1x PBS, pH

7.4) was then incubated for 30 min at room temperature to prevent the non-specific binding

on the SWCNTs [20]. After rinsing with PBS buffer (pH 7.4, 1x), OPN (1 pg mL-1 –1 µg

mL-1) in PBS (pH 7.4, 1x), undiluted human serum, and 10-fold diluted human serum with

PBS was applied to measure the resistance change of the immunosensors. During each

functionalization, all solutions were pipetted through a polydimethylsiloxane (PDMS) well

fabricated on the channel area, and all the immunosensors were rinsed with PBS buffer (pH

7.4, 1x) after each functionalization step. Fig. 1a shows a schematic of the SWCNT

immunosensor.

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2.4 Electrical measurements

After each functionalization step, all the immunosensors were thoroughly rinsed

with PBS buffer (pH 7.4, 1x) and dried at room temperature. The current vs voltage (I-V)

characteristics, or the electrical resistance, of all the immunosensors were measured using a

Keithley 2400 semiconductor characterization system, where voltage was varied from −2.0

to 2.0 V. The resistance of the SWCNT channels increased due to each functionalization.

The relative resistance change (RRC) is defined as ΔR (= R - R0)/R0, where R is the

immunosensor resistance after the attachment of OPN, and R0 is the immunosensor

resistance after the treatment of OPN antibody and Tween 20.

2.5 Non-electrical measurements: atomic force microscopy (AFM), scanning electron

microcopy (SEM), optical transmission, and X-ray photoelectron spectroscopy (XPS)

An atomic force microscope (Bruker Instrument, USA) and a field emission

scanning electron microscope (FE-SEM) (S-4800, Hitachi, Japan) were used to characterize

the surface morphology of the SWCNT channels and OPN antibodies on the channels.

AFM images were taken in the tapping mode using a V-shaped TESP probe (Bruker

Instrument). For FE-SEM images, samples were coated by a thin layer of platinum using

ion sputter (E-1045, Hitachi, Japan) at 20 mA for 60 sec before imaging. UV-Vis-NIR (Cary

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5000, Agilent, USA) was used to measure the optical transmission of the immunosensors.

The chemical composition of the SWCNT surface after DEP-deposition and the antibody

immobilization (10 µg mL-1) was studied by XPS (K–Alpha, Thermo Scientific, USA).

3. Results and Discussion

3.1 AFM, SEM, and XPS analyses

The SEM images of the SWCNT channels showed that many SWCNTs were

aligned between two Au/ITO electrodes by DEP (Fig. 1b and 1c). These aligned SWCNTs

contrast with a non-aligned and dense network of SWCNTs made by sedimentation [21].

The AFM images showed the deposition of the SWCNTs on a glass substrate (Fig. 1d) and

the OPN antibodies on the SWCNT surfaces (Fig. 1e). The spiky nanotube features

disappeared after the antibody attachment, and a globular surface of the antibody layer was

observed. The XPS measurements of the antibodies also showed that the antibodies were

successfully immobilized (supplementary text and Figs. S1-S3).

3.2 Electrical measurements after each functionalization and transparency measurements

Fig. 2 shows an I-V characteristic graph of the immunosensors (L- 2 µm and W-100

µm) when the OPN concentration was 1 pg mL-1. The I-V characteristic graphs for the other

channel lengths (L- 5 and 10 µm) are shown in the supplementary data (Fig S4). More than

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five immunosensors for each channel length were evaluated. The I-V graphs showed a

linear behavior after each functionalization for all the channel lengths, indicating a good

ohmic contact between SWCNTs and Au/ITO electrodes. The resistance of the

immunosensors increased with additional functionalization: EDC/NHS, OPN antibody,

Tween 20, and OPN. The average resistances (Re) of the immunosensors immediately after

the deposition of the SWCNTs were almost linear (R2=99.5%) with respect to the channel

length (L), showing Re = 1.18 × L, where Re and L are expressed in kΩ and µm, r

espectively (Fig. 3).

Deposition of a thin layer of gold with ITO resulted in a good ohmic contact

between the SWCNTs and the ITO electrodes as well as high transparency [22]. In fact, the

work functions of ITO and CNT are into the range of 3.9 - 4.4eV and 4.7 - 5.1eV,

respectively. This difference would show a large Schottky barrier at the source and drain

electrodes without the gold layer, which has the higher work function (5.1- 5.47eV). The

optical transmittance spectra of the immunosensors exhibited high transparency (~81%) in

the wavelength range of 300 ‒ 700 nm (visible light) while the immunosensors with only

ITO electrodes showed slightly higher transparency (~90%) (Fig. S5). Transparent

biosensors are considered attractive sensing platforms because they allow both optical and

electrical measurements in various experimental set-ups for biosensing and bio-MEMS [23,

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24].

To determine the optimal incubation time for OPN, the RRCs of the immunosensors

were measured with the incubation time for two different OPN concentrations (Fig. 4).

They drastically increased up to 20 min, and saturated after an incubation time of 60 min.

Therefore, the immunosensors were incubated for 60 min during all the experiments.

3.3 Sensitivity and selectivity studies

Fig. 5 shows the RRCs of the immunosensors as the OPN concentration in PBS was

varied from 1 pg mL-1 to 1 μg mL-1. The RRCs increased with an increase in the OPN

concentration, which is due to a decrease in the number of hole carriers of the p-type

semiconducting SWCNTs with attachment of negatively charged OPNs. For repeatability,

more than five immunosensors were tested for each concentration of OPN, which

confirmed that the immunosensor responses were reproducible. We also observed a highly

linear behavior of RRCs with the logarithm of OPN concentration for different channel

length (2, 5, and 10 µm), where the smallest R2 was 98% for 10 µm long channels.

The RRCs of the immunosensors were also measured as the OPN concentration in

10-fold diluted human serum and undiluted human serum was varied from 1 pg mL-1 to 1

μg mL-1 for 2 µm long channels (Fig. 6). The OPN concentration in prostate and normal

patient sera was reported to be 653±39 ng mL-1 and 439±30 ng mL-1, respectively [12].

13

Also, the average level of free OPN in plasma was 45±20 ng mL-1 in normal samples and

127±80 ng mL-1 in prostate cancer samples [13]. The RRCs for given OPN concentration

were the smallest in a PBS medium and the largest in undiluted human serum, which can be

attributed to non-specific binding of several other proteins in the serum.

To investigate the selectivity of the immunosensors, PBS (pH 7.4, 1x), 10-fold

diluted human serum, and undiluted human serum along with high concentration BSA (10

µg mL-1) in the three media were introduced to the immunosensors (Table 1). There was

little change in the RRC of the immunosensors between the media and BSA. However,

upon the addition of OPN (1 pg mL-1) on the immunosensors, a rapid and sharp change in

RRC was observed. According to the measurements, these immunosensors showed high

selectivity for OPN, and the detection limit for OPN against BSA and the media was found

to be 0.3 pg mL-1 when the three interpolated graphs in Fig. 6 are equated with the noise

levels caused by the media in the immunosensors, corresponding to 1.5 fg mL-1 for 1 μm2 of

the sensing areas. This immunosensor is 3 orders of magnitude more sensitive than the

current clinical method, ELISA immunoassay [25], and 4-5 orders of magnitude more

sensitive than the OPN levels detected in prostate and normal patient sera. This high

sensitivity shows several advantages. For example, it allows at least 1000-fold dilution of

real samples with buffers, in which less non-specific binding may occur and it is easier to

14

make electrical or magnetic manipulations for the concentration, capture, or detection of

target proteins.

A CVD-grown CNT-based biosensor demonstrated the detection limit of 1 pg mL-1

for OPN in PBS using a single chain variable fragment antibody [11], and a homogeneous

electrochemical method showed a detection limit of 10.7 ng mL-1 in a linear range from 50

to 500 ng mL-1 [26]. The previous prostate cancer immunosensors along with the present

study are listed in Table 2. According to the table, this immunosensor is a cost-effective,

highly sensitive and selective, easy to implement and transparent sensing platform allowing

for optical measurements. Moreover, this immunosensor involved the conventional

microelectrode fabrication and the room-temperature deposition of SWCNTs, implying that

this immunosensor can also be implemented on a flexible substrate for more applications.

4. Conclusions

We presented a label-free, highly sensitive and transparent electrical immunosensors

based on SWCNTs to detect a prostate cancer biomarker OPN. The SWCNTs were

deposited between two transparent Au/ITO electrodes on a glass substrate by DEP. We

observed a highly linear and reproducible behavior over a wide range of OPN

concentrations (1 pg mL-1 to 1 µg mL-1) in PBS and human serum along with a detection

limit of 0.3 pg mL-1. These immunosensors also demonstrated high selectivity of OPN

15

against BSA, human serum and PBS. This transparent immunosensor has great potential as

a useful platform for sensing different types of protein biomarkers along with simultaneous

optical measurements.

Acknowledgments

This research was supported by Basic Science Research Program through the

National Research Foundation of Korea (NRF) funded by the Ministry of Education,

Science and Technology (2012R1A2A2A01012528).

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Figure 1 (a) Schematic of the transparent SWCNT based immunosensors for OPN

detection, where OPN antibodies were immobilized on the SWCNT surface

between the source and the drain electrodes on a glass substrate, (b) FE-SEM

micrographs of SWCNTs between Au/ITO electrodes, (c) FE-SEM micrographs of

SWCNTs at higher magnification, (d) AFM images of the SWCNTs deposited

between two Au/ITO electrodes, and (e) AFM images of OPN antibody covalently

attached to –COOH functionalized SWCNT surfaces.

22

Figure 2 I-V measurements of the immunosensors (L- 2 µm and W- 100 µm) modified with

EDC/NHS, OPN antibody, Tween 20 and OPN antigen (1 pg mL-1).

Figure 3 Average resistances of the immunosensors after the deposition of the SWCNTs by

dielectrophoresis for various channel lengths (L-2, 5, and 10 µm).

23

Figure 4 Relative resistance changes of the immunosensors as the OPN incubation time

was varied from 10 to 80 min for different concentrations of OPN. The error bars

indicate the standard deviations of the measurements.

Figure 5 Relative resistance change of the immunosensors as the concentration of OPN in

PBS (pH 7.4, 1x) was varied from 1 pg mL-1 to 1 μg mL-1 for various channel

lengths (L-2, 5, and 10 µm), and constant channel width (W- 100 µm). Y and x

represent the relative resistance change of the immunosensors and OPN

concentration in g mL-1, respectively. The error bars indicate the standard

deviations of the measurements.

24

Figure 6 Relative resistance change of the immunosensors (L- 2 µm and W- 100 µm) as the

concentration of OPN in PBS (pH 7.4, 1x), 10-fold diluted human serum and

undiluted human serum was varied from 1 pg mL-1 to 1 μg mL-1. Y and x represent

the relative resistance change of the immunosensors and OPN concentration in g

mL-1, respectively. The error bars indicate the standard deviations of the

measurements.

25

Table 1 The selectivity test of the SWCNT immunosensor (L- 2 µm and W- 100 µm) in

response to PBS (pH 7.4, 1x), 10-fold diluted human serum, undiluted human

serum, and BSA (10 μg mL-1) and OPN (1 and 500 pg mL-1) in the three media.

Relative resistance changes were shown along with the standard deviations in the

parentheses.

Table 2 Comparison of previous prostate cancer immunosensors with the present study.