Selective and easy detection of microcystin-LR in freshwater using a bioactivated sensor based on multiwalled carbon nanotubes on filter paper

Highlights

• Inexpensive, simple, and sensitive paper sensor for Microcystin-LR (MC-LR) detection.

• MC-LR targeting aptamer (DNA oligonucleotide) - activated multi-wall carbon nanotubes biosensorl.

• Acceptable limit of detection (0.19 ng/mL) for drinking water.

• High selectivity to MC-LR among other MC congeners.

• A special advantage to use in remoted location.

Abstract

Microcystin-LR (MC-LR) is a cyanobacterial toxin produced as a result of eutrophication in polluted water in warm weather conditions. The MC-LR could cause health problems in mammal organs such as the liver, heart, and muscle. Therefore, the World Health Organization (WHO) has stipulated a limit of <1.0 ng/mL in drinking water. Thus, detection and quantification are vital, but current techniques require complex and expensive offsite analysis. We have developed an inexpensive, sensitive, and field-deployable sensor based on bioactivated multiwalled carbon nanotubes (MWCNTs, diameter 20 nm) and micropore filter paper (0.45-μm pore size) for the detection of MC-LR. A specially designed DNA oligonucleotide (5-NH₂-C₆-AN₆) was used as the MC-LR targeting aptamer (MCTA). For bioactivation, MCTA was immobilized on the carboxylated MWCNTs via the formation of amide bonds. The bioactivated MWCNTs were deposited on the micropore filter paper by suction filtering. The detection of MC-LR in freshwater was possible within 1.5 h, achieved by measuring the changes in electrical resistance caused by the selective MC-LR and MCTA interactions. Despite suffering from some matrix effects, the detection limit of the sensor was 0.19 ng/mL for low-concentration MC-LR (≤0.5 ng/mL). This method is much cheaper (biosensor price: < $2.5) than liquid chromatography coupled with tandem mass spectroscopy analysis (ca. $50/sample) which is a standard method for MC-LR detection in a modern laboratory. Thus, this cheap and straightforward MC-LR sensor has applications for detection in remote locations.

Introduction

Cyanobacterial outbreaks occur in surface water bodies as a result of eutrophication and warm weather. Cyanobacteria release a group of toxins known as microcystins (MCs) (Greer et al., 2018; USEPA, 2015). Of these, microcystin-LR (MC-LR) is a cyclic heptapeptide that consists of five non-proteinogenic amino acids and leucine (L) and arginine (R) residues at the two and four positions. MC-LR is the most toxic congener of the MCs, having a median lethal dose (LD50) of 43 μg/kg (Alosman et al., 2020). The World Health Organization (WHO) stipulates that the level of MC-LR in drinking water should be less than or equal to 1 ng/mL (WHO, 2003).

Currently, the MC-LR concentration in contaminated water is determined by high-performance liquid chromatography and tandem mass spectrometry (LC/MS/MS) (Draper et al., 2013; Picardo et al., 2019; Roy-Lachapelle et al., 2019). However, this technique cannot be carried out at sites of pollution because it requires sophisticated instruments. In addition, LC/MS/MS analysis involves high running costs ($50/sample in Korea) and is time-consuming. Several alternative MC-LR quantification methods have been reported but still require much improvement for practical applications (Picardo et al., 2019).

As part of the efforts to develop better biosensors (Li et al., 2019; Aliakbarinodehi et al., 2016; Taghdisi et al., 2017), carbon nanotube (CNT)-based biological sensing systems have been used. CNTs have attracted research interests as active elements for biosensors because of their high aspect ratio, large surface area, good electrical conductivity, and high chemical stability (Yola et al., 2021). In these sensors, detection is based on electrochemical methods. Examples include ionic liquid-CNT-modified electrodes (Aliakbarinodehi et al., 2016), electrochemiluminescent systems containing CNTs (Taghdisi et al., 2017), and electronic methods based on CNT field-effect transistors (FET), for which the latter has reached an MC-LR detection limit of approximately 0.6 ng/L (Tan et al., 2015). Nevertheless, these biosensors still have several disadvantages, including high cost, complexity, and the need for laboratory-based experiments.

A few years ago, our group reported (Ji et al., 2018) that bioactivated multi-wall carbon nanotubes (MWCNTs) deposited on a micropore filter paper could be used to detect prostate-specific antigen (PSA) at diagnostic levels (detection limit: 1.18 ng/mL) within 2 h. The detection mechanism of the MWCNT sensor is based on the change in the potential barrier between the p-type conducting filaments (MWCNTs) induced by the interaction between the target molecule (PSA) and active site (PSA antibody) attached to the MWCNTs. However, the drawback of this paper-based sensor is the lack of repeatability, which is mainly due to the uneven deposition of the MWCNTs on the filter paper and the instability of the attached PSA antibody. Specifically, the sensor could not be dried after the bioactivation process because the PSA antibody lost its activity, and the production yield was only around 30 %. Therefore, improvements in these paper-based sensors should be made to enable their practical application. Jin et al. reported (Jin et al., 2020) a sensor that allowed the diagnosis of early-stage pancreatic cancer by detecting CA19-9, a biomarker of this disease, using an improved device to detect the paper-based sensor. Their sensor could also be stored for up to 7 days by using a freeze-drying method.

Aptamers are synthetic single-stranded antibody-like DNA or RNA molecules that bind selectively to their target biomolecules (Aliakbarinodehi et al., 2017; Odeh et al., 2020). For the detection of target proteins, aptamers have significant advantages over antibody-based approaches, including easy synthesis, easy chemical modification, high stability, and reversible denaturation (Morales et al., 2018); most importantly, the activity of the dried aptamer is fully recovered upon re-wetting (Liu et al., 2011). In a recent report (Cunha et al., 2018), the MC-LR targeting aptamer (MCTA: DNA oligonucleotide 5-NH₂-C₆-AN₆) was selected from random libraries of synthetic DNA/RNA sequences using a SELEX selection procedure.

In this study, we present a reliable paper-based aptasensor for the label-free detection of MC-LR in freshwater. The immobilization of MCTA on MWCNTs achieved high sensitivity and specificity, and the MCTA-modified MWCNTs were evenly deposited on the micropore filter paper by suction filtering. We tested the produced biosensor for its detection sensitivity and selectivity under the matrix effect conditions related to the ions present in freshwater samples. The demonstrated strategy provides an inexpensive tool for the fast analysis of MC-LR in environmental samples at remote sites of undeveloped countries.