An Aptamer-Based Colorimetric Assay Integrated a Portable Spectrometer for on-Site Detection of Pbtx-2
Authors: Kong Liubing , Ma Chiyu , Wang Xinyi , Zhu Yuxuan , Kirsanov Dmitry , Legin Andrey , Wan Hao , Wang Ping 
Published: July 2021

Introduction

Brevetoxins (named using a notation system PbTx 1-n) produced by the algae species Karenia brevis have been increasing in geographical distribution [1]. Humans’ exposure to brevetoxins can occur through consumption of contaminated shellfish as well as aerosol exposure, causing a series of adverse symptoms clinically described as neurologic shellfish poisoning (NSP) including cramps, diarrhea, and coma, etc. Current methods for brevetoxin detection such as mouse bioassay (MBA), high-performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA), etc., feature the advantages of high sensitivity and reliability [2]. However, the dependence on bulk volume laboratory equipment and complicated pretreatment limit the widespread application in on-site brevetoxins detection. Therefore, a sensitive and efficient method for on-site determination of brevetoxin in contaminated shellfish is of great necessity.

In this work, an aptamer-based colorimetric assay is designed for sensitive detection of PbTx-2 which is the most predominant form of brevetoxins. The concentration of PbTx-2 can be easily transformed to the absorbance change which can either be directly visualized by a color change or can be quantified using a portable spectrometer. This approach provides an efficient way for PbTx-2 on-site detection combining the low cost and sensitivity of aptamer and the portability of self-designed spectrometer.

Method

The self-designed portable spectrometer (SDPS) is composed of a spectrometer sensor, a broad-band LED module (wavelength range of 470-850 nm), a micro-control module, and a communication module to contact with portable devices e.g., a personal computer (PC) (Fig.1 B, C). Optic fibers were used for guiding light emitted from the LED or reflected from the sample to reduce the volume of SDPS.

Aptamer BT10 was synthesized with high affinity to PbTx-2 according to published literature and it was heat-treatment at 92 ℃ for 10 min, followed by snap-chilling on ice for 5min and bringing it back to the room temperature to get an appropriate secondary structure [3]. Tyrosine-capped AuNPs were synthesized using tyrosine amino acid as a reducing and capping agent. 10 µL BT10 with a concentration of 0.3 µM was incubated with 20 µL different concentrations of PbTx-2 for 30 min to ensure complete binding. Then, 100 µL AuNPs solution was added into the above mixture for 5 min followed by 5 µL positively charged 3,3',5,5'-tetramethylbenzidine (TMB) for 5 min to induce aggregation of AuNPs. Therefore, free aptamers can be adsorbed on the surface of AuNPs and prevent the AuNPs from aggregation while the aptamer binding to PbTx-2 cannot (Fig.1 A). All reactions were performed in a solution chamber and finally the chamber was placed into the SDPS. Through signal processing on a PC, the concentration of PbTx-2 can be determined.

Results and Conclusions

From the spectrum results, the absorbance at 520 nm is decreasing while the absorbance at 610 nm is increasing with the concentration of PbTx-2 increasing (Fig.1 D). The ratio of absorbance at 610 nm and 520 nm is linear to the concentration of PbTx-2 with a linear range of 0.1 ppm to 10 ppm (Fig.1 E). Moreover, tyrosine-capped instead of citrate-capped AuNPs were chosen for colorimetric assay for experiment results show PbTx-2 can interact with citrate-capped not the tyrosine-capped AuNPs. The positively charged TMB was utilized for inducing AuNPs aggregation not the traditional sodium solution, for we find TMB can be more sensitive for aptamer number change on the surface of AuNPs. This approach allowed a limit of detection of 0.05 ppm PbTx-2 within a detection time of 40 min. Compared with other methods for PbTx-2 detection, this approach features portability and cost-effectiveness, which can be used in resource-limited areas.

References

[1] S.M. Watkins, A. Reich, L.E. Fleming, R. Hammond, Neurotoxic shellfish poisoning, Mar Drugs, 6 (2008) 431-455.

[2] J. Naar, A. Bourdelais, C. Tomas, J. Kubanek, P.L. Whitney, L. Flewelling, K. Steidinger, J. Lancaster, D.G. Baden, A competitive ELISA to detect brevetoxins from Karenia brevis (formerly Gymnodinium breve) in seawater, shellfish, and mammalian body fluid, Environ Health Persp, 110 (2002) 179-185.

[3] S. Eissa, M. Siaj, M. Zourob, Aptamer-based competitive electrochemical biosensor for brevetoxin-2, Biosens Bioelectron, 69 (2015) 148-154.

Figure 1