Label-free fluorescent aptasensing of mycotoxins via aggregation-induced emission dye

Highlights

• A label-free aptasensor for mycotoxin was built via aggregation-induced emission dye.

• The aptasensor achieved mycotoxin detection by the use of single-aptamer sequence.

• The aptasensor may sever as facile platforms for food and environmental analysis.

Abstract

Aggregation-induced emission (AIE) dyes now serve as powerful tools for fluorescence analysis and biological imaging due to their light-up property in the aggregated state. Herein, we proposed an AIE dye-based aptasensor, enabling label-free and turn-on detection of mycotoxin. The positively charged AIE dyes would confer high-affinity to negatively charged aptamers and be lighted up by the aggregation process of dye molecules. Attributed to the resistance of ochratoxin A (OTA)-binding aptamer complex to enzyme digestion, label-free analysis of OTA could be realized via the synergetic utilization of AIE dyes and enzymatic digestion process. The digestion process could contribute to a signal to background ratio (S/B) of 1.71 for OTA detection. Remarkably, the adoption of AIE dyes would lead to a dramatic enhancement of over 104.5% of S/B compared to conventional nucleic acid dyes, thus favoring the sensitivity of the aptasensor. Only one aptamer sequence was involved in the assay, thus avoiding the complex structure design of aptamer probes. The AIE dye-based aptasensor could confer a limit of detection of 0.4 ng/mL for OTA detection. Besides, it exerted remarkable specificity for identifying OTA, and has been successfully applied for OTA analysis in wine and coffee. AIE dyes would be a compelling candidate in biohazard screening filed, and the proposed aptasensor would potentially provide a universal platform for on-site detection of food and environment contaminations.

Introduction

Aggregation-induced emission (AIE) is a photophysical phenomenon in which a group of fluorescent dyes glows weakly in the dilute solution state, while their fluorescence is significantly enhanced in the aggregation state [[1], [2], [3], [4]]. The restriction of their intramolecular rotations in the aggregate state may contribute to the strong fluorescence. Hence, AIE dyes could settle the dilemma of concentration quenching or aggregation caused quenching (ACQ) of conventional dyes or fluorophores. Typical AIE dyes are 9,10-distyrylanthracene (DSA), tetraphenylethene (TPE), silacyclopentadiene (silole), and their derivatives [5]. These fluorescent dyes with AIE properties have showed prominent potential in the development of fluorescent biosensors and bioimaging [[6], [7], [8], [9], [10], [11], [12], [13]]. Especially, AIE dyes with positive charges was already testified to be able to strongly bind to DNA strands [5]. On this base, AIE dyes may potentially serve as powerful tools for constructing label-free and turn-on nucleic acid probes.

Aptamers are man-made single-stranded oligonucleotides, harboring high specificity and affinity towards various target species [[14], [15], [16], [17]]. The utilization of aptamers can act as a substitute for antibodies to construct a common detection platform [[18], [19], [20], [21], [22], [23]]. In particular, aptamers possess some novel features, such as superior controllability, high stability and easy to synthesize [[24], [25], [26], [27]]. Enormous aptamer probes have been designed to recognize proteins, antibiotics, viruses or mycotoxins [[28], [29], [30], [31], [32], [33], [34]]. Profited from the inherently nucleic acid nature [24,35,36], the strategies of these aptasensors are usually based on the structure-switching process of probes upon target binding [[37], [38], [39], [40], [41]]. Paradigms of structure-switching probes include aptamer molecular beacons, displacement probes and splitting aptamer probes [42]. Through the mechanism of structure-switching process, these probes adopt binding-induced structural change to specifically and rapidly output the signals [37,[42], [43], [44]]. Yet, limited by the intrinsic complex structure of aptamers, the design of the structure-switching aptasensors, sometimes, is of considerable difficulty [24,29,42,45]. Otherwise, the stability of the nucleic acid structures also needs to be elaborately optimized to obtain an efficient response to target molecules [42,46]. Apparently, the utilization of intrinsic response of aptamers to targets would obviate the complex structure design, and allowing to construct aptamer probes by the direct use of aptamer sequence.

Motivated by the features of AIE dyes and aptamers, we proposed a label-free, turn-on aptasensor by the direct use of aptamer sequences. As a proof of concept, ochratoxin A (OTA) was chosen as the target. OTA is recognized as one of the most critical issues of pollution, and is also the key component of mycotoxins [47]. Excessive OTA may exert a serious threat to the human health. Among the AIE dyes, the cationic DSA derivative (with short alkyl chain, DSAI) would confer a high turn-on ratio upon binding to nucleic acids [5]. Besides, the excellent water solubility and highly charged state would further facilitate its use as fluorescent indicator for nucleic acid probes [48]. And we have successfully used DSAI for labeling a structure-switching “turn-off”aptamer probe [41]. Thus, DSAI dyes were chosen for construction of the aptasensors. The label-free and turn-on analysis of OTA could be realized via the synergetic utilization of AIE dyes and enzymatic digestion process. Further, contributed to the specificity of aptamers, this aptasensor could strictly identify OTA from other analogues. Notably, this label-free design strategy could contribute to a cheap, convenient, easy-operation and homogeneous assay for OTA detection. The appealing features of this design could render AIE dye-based aptasensors capable for on-site detection, thus serving as a potential platform for food safety control and environmental monitoring.