Small molecules such as antibiotics, drugs, toxins and food additives are often perceived as difficult targets to obtain a suitable affinity ligand. The development of rapid, cost-effective, single-step assays for the sensitive and specific detection of small molecules are very attractive for many applications ranging from life sciences to environmental analysis. Unfortunately, current methodologies still require multiple complex, time consuming washing and incubation steps, which limit their applicability. Other issues such as lack of suitable antibodies and their relatively low signalling output add to these limitations. Therefore, tools to detect and quantify small molecules directly in crude samples are in high demand.
The solution – Aptamers
Nucleic acid aptamers offer an alternative means of recognising small molecules. They have been demonstrated as an efficient and viable alternative to overcome the above limitations because of their increased affinity and specificity towards their target. Unlike antibodies, aptamers are devoid of a rigid backbone structure. This means that aptamers are able to form complex folds and cage their targets, allowing them to form tighter and more specific interactions (Figure 1).
Figure 1. Schematic representation of an aptamer binding specifically to its small molecule target.
Our approach for detecting small molecules
At Aptamer Group Limited (AGL), we select aptamers that bind to small molecules with high specificity using our specially designed proprietary automated displacement approach. This method allows us to select aptamers that can be readily utilised in various biosensor platforms for the detection of small molecules without the need for ‘sandwich-pairs’ or a ‘competitive assay’ format. Conformation change / displacement of the aptamers upon small molecule binding, gives a ‘signal amplification’ effect also termed as ‘gain of signal’ which can be used as a basis for small molecule detection. This allows simplified downstream assay development and makes them useful as alternatives such as ELISA-like assays, Lateral Flow Devices (LFD’s), Molecular Beacons and Biosensors.
This is what our customers say
Our customers feedback is very important to us. They help us to constantly improve our overall service to our customers all over the world. With the help of our dedicated researchers, we have successfully completed multiple small molecule projects in their agreed timeframes. Figure 2 is an example of the feedback we have got from one of our satisfied customers.
Figure 2. Customer feedback on our overall service.
Displacement Case Study
The following data demonstrates aptamer discrimination between similar small molecules, distinguishing a chemotherapeutic from its metabolite using BioLayer Interferometry (BLI) and ELISA-like assay (fluorescent plate reader assay). Results from both assays demonstrate that the aptamer binds specifically to the chemotherapeutic target compared to the metabolite competitors and the negative target.
Small Molecule detection using our displacement approach via BLI
Aptamers are readily immobilised onto commercially available biosensor platforms, including BLI, using a simple ‘Dip and Read’ biosensor format. Using this principle, we have demonstrated that our aptamers are able to distinguish a chemotherapeutic target and its metabolite, as well as its negative target (Figure 3).
Figure 3. Binding of the aptamer to the chemotherapeutic target (red trace), a key metabolite (yellow trace) and the negative target (blue trace) using BLI.
Small molecule detection using our displacement approach via an ELISA-like assay
The following data demonstrates concentration-dependent binding of the aptamer to its target using an ELISA-like assay. The results were comparable with BLI assay showing increased aptamer binding to the target as compared to its metabolite and the negative target (Figure 4).
Figure 4. Binding of the aptamer to the chemotherapeutic target, a key metabolite and the negative target using an ELISA-like assay.
Small molecule detection in plasma via an ELISA-like assay
As with other Aptamer Group selection methodologies, we isolate and design our small molecule targeting aptamers using selection conditions tailored to the end application. In this project, a key consideration was the final matrix utilised in the end assay (buffered plasma). To confirm that the end aptamers were tailored to the end use, concentration dependant binding was observed in several concentrations of buffered plasma (0, 10, 20 and 25%) using an ELISA-like assay (Figure 5).
Figure 5. Binding of the aptamer to the chemotherapeutic target in different plasma concentrations using an ELISA-like assay.
Overall, the data suggests that our aptamers can be readily incorporated into BLI assay and existing ELISA platforms, and show target specific responses even in crude sample matrices. At AGL, we have developed and validated numerous such small molecule specific aptamers in many similar biosensor and assay formats. These aptamers can be used in a rapid, gain of signal assay that can be used to discover, deduce, and quantify sample compounds that can reduce the heavy reliance on antibodies and mass spectrometry. To know more about our small molecule aptamer detection methods, please contact us using the form below.