App note

Reversible Aptamer Sensing of a Small-Molecule Drug

Small drugs give small SPR signals. Splitting an aptamer in two makes the signal bigger and lets the sensor follow the drug up and down, with no regeneration step.

SystemP4SPR

ApplicationTherapeutic drug monitoring, aptamer design

SampleBuffer, cerebrospinal fluid, diluted plasma

In short: Researchers at the BioMed X Institute in Heidelberg used the P4SPR to design a reversible sensor for vancomycin, an antibiotic with a narrow therapeutic window. They found the one split site that works out of three, showed that the signal rises and falls with the drug within seconds, and ran the sensor for nine hours in a fluid as complex as cerebrospinal fluid.

The need

Vancomycin treats serious infections, including MRSA and brain infections after surgery, but the window between too little and too much is narrow, and patients differ widely. Drug levels are measured today by lab immunoassays or mass spectrometry, so samples are few and results come late. A sensor that follows the drug continuously would need to do two things SPR finds hard: see a small molecule (1.4 kDa), and let go of it when the level drops.

How it works

An aptamer, a short DNA strand that binds vancomycin, is cut in two. One half is anchored on the sensor; the other half is in the sample. Only when vancomycin is present do the two halves and the drug lock together on the surface, so the signal comes from a whole DNA strand rather than from the small drug alone. When the drug is washed away, the complex falls apart and the sensor is back where it started (Figure 1).

Half aptamers on the sensor; with the drug, the second half and the drug lock on; without it, they leave and the sensor resets — Figure 1. The split-aptamer sandwich: half an aptamer on the sensor; the drug and the second half lock on together; when the drug is gone, they leave and the sensor resets.

What the study showed on the P4SPR

	Result
Design screening	Three split sites compared side by side: one kept vancomycin binding, two did not
Reversibility	Binding and release within seconds; repeated doses gave the same response each time (Figure 2)
Robustness	Same response across physiological pH and magnesium and calcium levels; similar performance in artificial cerebrospinal fluid and in buffer
Complex samples	Worked in dog cerebrospinal fluid, and for 9 hours of repeated measurements in diluted rat plasma with almost no baseline drift

Illustration of a reversible readout: responses to 10, 50 and 100 micromolar vancomycin, then repeated 50 micromolar doses, each returning to baseline — Figure 2. A reversible readout (illustration): rising doses of vancomycin (10, 50 and 100 µM), then the same 50 µM dose repeated. Each wash brings the signal back to baseline, with no regeneration step.

Full method and data: Santa et al., Analyst 2025 (reference below).

Why the P4SPR fits aptamer development

What the lab needs	What the P4SPR brings
Compare designs quickly	Four channels, read in turn: several aptamer variants, or sample and reference, on one sensor
See whether binding is reversible	Real-time signal: binding and release are visible directly
Test at body temperature	Measurements at 37 °C
Oriented, reusable DNA surfaces	Biotinylated aptamers captured on a streptavidin layer
Work in biological fluids	AffiCoat low-fouling surface available for protein-rich samples

Beyond vancomycin

The split-aptamer approach applies to any small molecule with a known aptamer: other antibiotics, anticancer drugs, neurotransmitters or metabolites. The P4SPR is a quick way to try split sites and stem lengths before building a dedicated sensor. For DNA surfaces in general, see our note on DNA hybridization.

What you need

Item	Source
P4SPR	Affinité Instruments
Biotin Sensor (SAMBT-01) and Streptavidin Kit (STRP-KIT)	Affinité Instruments
Split-aptamer pair: one half biotinylated at its 5′ end, the other free	Custom DNA synthesis (sequences in the reference)
Running buffer with magnesium; artificial cerebrospinal fluid if needed	Standard reagents

Reference

C. Santa, S. Park, A. Gejt, H. A. Clark, B. Hengerer, K. Sergelen, "Real-time monitoring of vancomycin using a split-aptamer surface plasmon resonance biosensor," Analyst, vol. 150, pp. 131–141, 2025. doi:10.1039/d4an01226g (open access).

Illustrations by Affinité Instruments. Figure 2 illustrates the measurement pattern described in the reference above; it is not measured data.