Infectious diseases are among the common leading causes of morbidity and mortality worldwide. Let’s take an example of the current scenario where the world is gripped by the novel highly pathogenic COVID-19 virus with 4,305,477 confirmed cases and 289,867 deaths worldwide as of 13th May 2020 (Source-WHO). Associated with the emergence of new infectious diseases, the increasing number of antimicrobial resistant pathogens presents a serious threat to public health and hospitalized patients.

Toxins are one such pathogen that are biological in origin and secreted by bacteria, fungi, plants or animals. Some have mechanisms of action and physical properties that make them amenable as infectious and potential bioterror warfare agents due to their short incubation period and high toxicity. They are broadly categorized as follows:

  • Bacteria: creates endotoxins, exotoxins, and enterotoxins
  • Fungi: create mycotoxins
  • Algae: create phycotoxins
  • Plants: create phytotoxins
  • Animals: create zootoxins

Long-term consequences of these toxins include effects on the immune, reproductive or nervous system, and also cause cancer. Because of such toxic properties, the presence of toxins in food and water has been of great concern in many countries. Detection systems that can reveal the presence and identity of these pollutants in clinical fluids, environmental samples, food and water systems are essential for guaranteeing heath and safety. Therefore, a permanent demand for appropriate detection methods those are affordable, practical, careful, rapid, sensitive, efficient and economical are needed.

Conventional chromatography techniques and immunoassays have been the used as detection systems but they are still plagued by:

  • Expensive and highly sophisticated instrumentation
  • Lengthy and complex preparation techniques and analysis
  • pH and temperature sensitive antibodies making them highly susceptible to denaturation.

More recent research focus has thus shifted to the development of DNA aptamer biosensors as a feasible alternative in detecting food and water contaminants at very low concentrations. Aptamers have increasingly found their way into analytics where they are used to replace antibodies because of their following applications such as:

  • Easy to synthesize and quick to screen
  • Can be selected in different environmental conditions
  • Selected aptamers are capable of adapting unique tertiary structures
  • Recognize target molecules with high affinity and selectivity
  • Can be modified chemically at defined positions and linked stably to solid surfaces
  • Can be reproducibly obtained in large quantities and at a low cost

Taking advantage of highly specific aptamers, two independent researchers have demonstrated aptamer-based methods for detecting toxins in bacterial and fungal pathogens.

Aptamer based LFA for rapid and sensitive detection of Cholera toxin

Cholera toxin has been responsible for the outbreak of cholera disease, a severe diarrheal illness, which has been a threat to thousands of lives in less developed countries. It is a bacterial enterotoxin that is transmitted by contaminated food or water. Frohnmeyer et al., 2018 employed a novel combination of capture aptamer against cholera toxin and GM1-functionalised liposomes to develop a very sensitive and antibody free sandwich LFA for cholera toxin detection.

The test line consisted of biotinylated aptamer CT916 pre-incubated with NeutrAvidin for the capture. The test was run sequentially with Cholera toxin B subunit (CT-B) spiked buffer at concentrations ranging from 0.1 to 10000 ng/ml and liposomes in buffer, allowing the immobilized aptamer to capture CT-B and subsequently GM1-lipsomes on the test line. The limit of detection of cholera toxin was 2 ng/ml or 10 ng/ml (visual) in 15 min (Figure 1A).

Figure 1. (A, left) Antibody free GM1 liposome LFA in the presence and absence of CT-B. (B, right) Sandwich LFA using aptamer-functionalized AuNPs and capture antibodies in the presence and absence of CT-B.

In addition, the authors established a semi-quantitative competitive and two sandwich type LFAs using aptamer-functionalized AuNPs and capture antibodies with even better detection limit of 0.6 to 51 ng/ml or 1 to 100 ng/ml (visual) with a 20 min total assay time (Figure 1B). These GM1-aptamr sandwich LFA tests provided an excellent alternative for cholera toxin detection in places where quick and reliable results are most needed along with proving that aptamers can serve as bio-recognition elements.

Aptamer based fluorometry and SERS assay for rapid and sensitive detection of fumonisin B1 toxin

Fumonisin B1 (FB1) is considered to be a major fungal mycotoxin with strong toxicity and is found in agricultural commodities, such as corn, wheat, and cereal products posing a global threat to food safety and public health. In a very recent publication, He et al., 2020 reported a dual mode counter propagating- responsive assay for the quantitative determination of FB1 by using fluorophore labeled aptamer as a probe and cDNA – modified AuNR as a quencher thus combining two ultrasensitive techniques namely, Surface – enhanced Raman scattering (SERS) and fluorometry. This assay allowed sensitive and reliable analysis by mixing the target sample, fluorescent aptamer probe and cDNA modified AuNR. In the absence of FB1, the aptamer and its cDNA would associate emitting strong SERS and weak fluorescence signals. In the presence of FB1, the aptamer disassociated with its cDNA and bound to the target with decreased SERS and increased fluorescence signals (Figure 1, top left). The limit of detection (LOD) of the fabricated assay was determined to be 3 pg/mL for SERS mode and 5 pg/mL for fluorescence mode based on signal-to-noise of 3 (S/N = 3).

Selective recognition of FB1 by the assay was verified by using six other kinds of mycotoxins, FB2, FB3, AFB1, ZEN, PAT, and OTA. The signal changes in the SERS and fluorescence intensities of the assays reflect their selectivity (Figure 2A and 2B). The assay was applied to the determination of FB1 contents of spiked corn samples that from 92 to 107%, confirming the practicality of this method. The results obtained by this assay are in good agreement with that of LC-MS/ MS method (Figure 2C). The dual determination provided reliable and accurate data with an inspiration to design similar other aptasensors thereby reducing the risk of false-positive or false-negative results.

Figure 2. (A and B) SERS and fluorescence intensities measured for the target FB1 and other mycotoxins with constant concentrations set to 10 pg/ml. (C) Recovery results for the added standard FB1 from corn samples.

Overall, aptamers offer a very promising approach to new generation detection protocols for toxins. The commercial applicability of aptamer kits is expected to improve over the coming years, especially with improvements in the selection and post-identification process of aptamer sequences. Aptamer based assay kits are expected to become a strong choice in the future detections of food and other toxins.

At Aptamer Group Ltd (AGL), we have developed our selection process tailored to customer requirements such as introducing specific buffers, matrices, and most importantly counter-selection steps to ensure that the aptamers are specific to the target of interest. In addition, similar biosensors have been developed for detecting toxins and other small molecules using our high affinity aptamers. If you would like more information on such platforms, please contact us using the form below.

References:

Frohnmeyer, E., Tuschel, N., Sitz, T., et al. (2019). Aptamer Lateral Flow Assays for Rapid and Sensitive Detection of Cholera Toxin. The Analyst. doi:10.1039/c8an01616j

He, D., Wu, Z., Cui, B. et al. (2020). Aptamer and gold nanorod–based fumonisin B1 assay using both fluorometry and SERS. Microchim Acta 187, 215 https://doi.org/10.1007/s00604-020-4192-0

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