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New paper: Novel aptamer biosensor for multiplex detection of small molecule targets

In the recently published paper in Talenta from Dr Mark Platt and the team at Loughborough University, they demonstrate a novel approach for aptamer biosensors to detect and quantify small molecule antibiotics and chemotherapeutic targets using aptamers from Aptamer Group.

This study highlights a simple method for integrating aptamers into RPS-based biosensors, with sensitive detection of multiplexed samples of small molecule targets.

This is an exciting advancement for our sensor methodologies. It shows how these components can be integrated to detect multiple small molecule targets. The signal we observe as the aptamer binds to the target, which is enhanced by the way the Aptamer Group have designed the Aptamers, opens up our methods to detect ANY target, regardless of the final tertiary stricture of the DNA.

Dr Mark Platt, Team Leader, Loughborough University

Here we have summarised the paper, discussing how these results can be used to improve small molecule aptamer biosensors.

What is Resistive Pulse Sensing?

Resistive pulse sensing (RPS) is a method based on the Coulter principle. This is a powerful method for particle counting and sizing in electrolyte solutions. Incorporating aptamers into these RPS biosensors has expanded the original DNA sequencing capabilities to be able to detect small molecules, proteins, pathogens, and metal ions.

To generate aptamer biosensors, a target-specific DNA aptamer I sbound to a nanocarrier particle. Each translocation of a nanocarrier through the nanopore produces a pulse on the RPS biosensor system. The pulse magnitude is relative to the volume of the nanocarrier, while the width of the pulse relates to its velocity.

Larger signals from greater conformational changes in the nanoparticle size and velocity mean that it is easier to measure binding using these aptamer biosensors. So, relying upon the conformational change that occurs when the aptamer binds to its target, particularly for small molecule targets, can limit the potential signal of aptamer biosensors.

In the new paper, the scientists use Aptamer Group aptamers to demonstrate a new approach that allows aptamers to be easily incorporated into the biosensor, regardless of the target affinity or resulting conformational change upon target binding. Rather than assess the target binding by conformational change of the aptamer on the nanocarrier surface, they analyzed the aptamer’s displacement from the nanocarrier particle as it preferentially binds to the target.


Developing the aptamer biosensor for small molecule targets

The researchers used three small molecule targets to develop the aptamer biosensor: the antibiotic moxifloxacin and two chemotherapeutics, imatinib and irinotecan.

Each of the aptamers was anchored onto the nanocarrier surface via a short ssDNA anchor sequence. Initial validation work carried out by Aptamer Group to develop the aptamers showed that upon binding the small molecule target, the aptamer undergoes a conformational change and dissociates from the surface. As the aptamers dissociate from the nanocarrier in the aptamer biosensor, the smaller aptamer-free particle’s velocity decreases.

Specific and sensitive small molecule detection with aptamer biosensor

In the moxifloxacin aptamer biosensor, incubation of the aptamer-loaded nanocarriers with increasing concentrations of the antibiotic over the range 0-200 µM showed a concomitant decrease in the velocity of the nanocarrier to base signal level. This correlation indicates that the aptamers dissociated from the nanocarrier by binding the target in a concentration-dependent manner.

The aptamers were shown to be specific for the small molecule drug, as no change in velocity was seen with incubation of a control antibiotic, ciprofloxacin.

Further calibration of the aptamer biosensor allowed quantification of lower target concentrations over the range 0.568-5.68 µM, demonstrating sensitive detection over an order of magnitude.

Similarly, for the chemotherapeutic targets, as the target concentration was increased (0-7 µM for imatinib, 0-13 µ for irinotecan), the carrier’s velocity decreased, showing the aptamer is being displaced from the surface.

Multiplexing small molecule detection

Multiplex analysis is possible in this aptamer biosensor format by utilizing different sized nanocarriers that can be distinguished by their relative velocity through the nanopore.

Using two different sizes of nanocarriers, 150 nm and 300 nm, Platt’s team functionalized them with the moxifloxacin and imatinib small molecule aptamers, respectively. The aptamers dissociated in the presence of their target with the aptamer biosensor able to show specific target detection. No dissociation occurred when incubated with the irinotecan control molecule, demonstrating the aptamers’ target specificity and the potential for multiplexed assays for rapid and sensitive analysis.

At Aptamer Group, we have developed and refined our specific processes for the selection of aptamers to small molecule targets. We have excellent success rates in developing aptamer and Optimer™ reagents to a range of small molecule targets. Get in touch to find out how we can help you with your next project.

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