Single-walled carbon nanotubes based quenching of free FAM-aptamer for selective determination of ochratoxin A

doi:10.1016/j.talanta.2011.08.015

Talanta

Volume 85, Issue 5, 15 October 2011, Pages 2517-2521

https://doi.org/10.1016/j.talanta.2011.08.015 Get rights and content

Abstract

Ochratoxin A, a toxin produced by Aspergillus ochraceus and Penicillium verrucosum, is one of the most abundant food-contaminating mycotoxins in the world. It has been classified by the International Agency for Research on Cancer (IARC) as a possible human carcinogen. In this paper, a sensitive and selective fluorescent aptasensor for ochratoxin A (OTA) detection was constructed, utilizing single-walled carbon nanotubes (SWNTs) as quencher which can quench the fluorescence of free unfolded toxin-specific aptamer attached with FAM (carboxyfluorescein). Without any coating materials as compared to graphene-oxide based sensor, we obtained the detection limit of our sensing platform based on SWNTs to be 24.1 nM with a linear detection range from 25 nM to 200 nM. This technique responded specifically to OTA without interference from other analogues (N-acetyl-l-phenylalanine, warfarin and OTB). It has also been verified for real sample application by testing 1% beer containing buffer solution spiked with a series of concentration of OTA.

Highlights

► Single-walled carbon nanotubes in combination with aptamer can detect ochratoxin A. ► Ochratoxin A induces the switching of the aptamer to antiparallel G-quadruplex. ► The G-quadruplex cannot be wrapped onto the single-walled carbon nanotubes. ► Low concentration of ochratoxin A can be detected without any coating materials.

Introduction

Nowadays, a variety of nanomaterials such as nanoparticles, nanotubes and nanowires have been used to create new types of analytical tools for life science and biotechnology [1]. Carbon nanotubes have emerged as one of the most extensively studied nanomaterials due to their unique chemical, electrical, and mechanical properties [2], [3]. The potential for application of carbon nanotubes ranges from molecular electronics to ultrasensitive biosensors. Compared to graphene-oxide which has flat surface, single-walled carbon nanotube has curved surface which may lower down the unspecific adsorption. In previous study [4], we have shown that uncoated graphene oxide has very poor sensitivity. Although coated graphene-oxide has significantly improved the detection limit, uncoated material which provides a simple approach and comparable detection limit is highly desirable.

The superpower of the above-mentioned carbon nanomaterials lies in that they can quench the fluorescence of the adsorbed dyes, which offers great opportunities for building a variety of biosensors especially when in combination with aptamer technology [5]. In recent years, aptamers as newly emerging molecular recognition agents have been extensively studied [6]. Aptamers are single strand DNA that can bind target molecule with high specificity and strong binding affinity [7], [8]. As compared to antibody, aptamer possesses several advantages which attract extensive research in recent years [5], [9], [10]: easily synthesized, commercially available, flexible modification with a variety of groups and good stability [11]. Until now, there are several reports which have demonstrated the powerful potential of biosensors based on combination of single-walled carbon nanotube and dye-modified DNA (or aptamer) [12], [13], [14], [15], [16]. Analytes such as ATP, cocaine, Hg²⁺ and DNA have been systematically investigated by using the above-mentioned approach. Among these experiments, the target analyte induced the DNA probes to be detached from single-walled carbon nanotube due to the strong binding between target and DNA probes. In our study, we will show that this sensing strategy can also be applied to detect target which has much weaker interaction with aptamer probes.

Recently, aptamer selected for specific binding with OTA has been used to construct different versions of biosensors. As compared to those methods based on toxin-specific antibody [17], [18], [19], [20], biosensors based on aptamer specific for OTA are highly flexible [4], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32]. Although in recent two years more and more papers come out about the detection of OTA by utilizing aptamer, those procedures and modification steps are still not simple and straightforward enough, which always need more than one DNA probe and more expensive labeling.

OTA, as one of the most abundant food-contaminating mycotoxins in the world [33], [34], was chosen as the prototypic analyte in this paper. Human exposure to OTA occurs mainly through consumption of improperly stored food products, particularly contaminated grain and pork products, as well as coffee and wine grapes [35]. This toxin has also been found in the body of animals, including human blood and breast milk [36]. OTA can cause cancer in human beings [37]. It has been shown to be weakly mutagenic and can cause immunosuppression as well as immunotoxicity [34], [38], [39]. With the recognition of severe toxic effect of this fungi toxin, a vast amount of study have been devoted to develop simple sensing platforms to detect this fungi toxin [40].

In this study, in combination with single-walled carbon nanotube, an aptamer specific for OTA was utilized, which can fold to form antiparallel G-quadruplex structure upon exposure to OTA [21], [41]. The formation of antiparallel G-quadruplex structure will prevent the adsorption onto the SWNTs. Coexistence of unfolded single-stranded DNA (ssDNA) and SWNTs will favor the spontaneous self-assembly in aqueous solution via hydrophobic driving force between DNA bases and SWNTs sidewall [42]. By employment of dye-labeled DNA as the recognition component, the strong interaction between ssDNA and SWNTs can bring the dye close to SWNTs, which, in turn, results in complete quenching [13]. Therefore we designed a sensing strategy for detection of OTA: FAM (carboxyfluorescein) modified aptamer after binding to OTA cannot be quenched by SWNTs, however, FAM modified aptamer without binding to OTA could be significantly quenched. Indeed, the results in our experiment also confirmed that our design strategy was very successful. By combining SWNTs with aptamer modified in single end, the major advantage is that it averts expensive dual labeling of aptamer in comparison to conventional molecular beacon. Furthermore, the unspecific adsorption of target molecules onto the single-walled carbon nanotubes based sensor is extremely low compared to the case for graphene oxide [4], which needed polymer coating.

Section snippets

Materials and reagents

OTA's aptamer (5′-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-FAM-3′) were synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Single-walled carbon nanotubes were purchased from Chengdu Organic Chemicals Co. Ltd. (Chengdu, China). DNA stock solution was prepared by dissolving oligonucleotides in 10 mM Tris buffer (pH 8.5) containing 120 mM NaCl and 5 mM KCl and 20 mM CaCl₂ and was stored at 4 °C before use. The concentration of oligonucleotide was quantified using the

Design strategy for ochratoxin detection

Scheme 1 illustrates the sensing strategy for detection of OTA. In the absence of target molecule (OTA), FAM-modified aptamer is wrapped around SWNT through π–π stacking interaction between the nucleotide bases and the SWNT sidewall to form stable complex [12], [43]. Consequently, the fluorescence of FAM is quenched readily via energy transfer from dye to carbon nanotube [13]. In the presence of target molecules (OTA), the conformation of the aptamer specific for OTA is switched from a random

Conclusions

To summarize, we have developed a simple, selective and sensitive fluorescent aptasensor for OTA detection by using a dye-labeled aptamer and SWNTs. A limit of detection of 24.1 nM was achieved for detection of OTA. The advantages of this analytical method rely on its simple measurement performance, allowing measurements to be performed almost in a real-time manner even at room temperature. The integration of nanostructures such as SWNTs with the use of specific recognition molecules such as

Acknowledgements

This work was supported by National Natural Science Foundation of China (Nos. 20905056 and 21075120), and 973 Project (2009CB930100 and 2010CB933600).

References (46)

L.F. Sheng et al.
Biosens. Bioelectron.
(2011)
L.B. Zhang et al.
Biosens. Bioelectron.
(2010)
B. Prieto-Simon et al.
Biosens. Bioelectron.
(2008)
B. van der Gaag et al.
Food Control
(2003)
L. Bonel et al.
Biosens. Bioelectron.
(2011)
L. Barthelmebs et al.
Food Control
(2011)
A. De Girolamo et al.
Food Chem.
(2011)
N. Duan et al.
Chin. J. Anal. Chem.
(2011)
H. Kuang et al.
Biosens. Bioelectron.
(2010)
C. Yang et al.
Biosens. Bioelectron.
(2011)

C. Juan et al.

Food Chem.

(2008)

M.M. Ngundi et al.

Biosens. Bioelectron.

(2006)

M.H. Yang et al.

Biosens. Bioelectron.

(2010)

C.M. Niemeyer et al.

Angew. Chem. Int. Ed.

(2001)

D. Tasis et al.

Chem. Rev.

(2006)

P.M. Ajayan

Chem. Rev.

(1999)

T. Hermann et al.

Science

(2000)

K. Sefah et al.

Nat. Protoc.

(2010)

A.D. Ellington et al.

Nature

(1990)

E.J. Cho et al.

Ann. Rev. Anal. Chem.

(2009)

M. Famulok et al.

Chem. Rev.

(2007)

I. Willner et al.

Angew. Chem. Int. Ed.

(2007)

K.S. Schmidt et al.

Nucleic Acids Res.

(2004)

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Single-walled carbon nanotubes based quenching of free FAM-aptamer for selective determination of ochratoxin A

Abstract

Highlights

Introduction

Section snippets

Materials and reagents

Design strategy for ochratoxin detection

Conclusions

Acknowledgements

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