Compact RNA sensors for increasingly complex functions of multiple inputs.

Designing single molecules that compute general functions of input molecular partners represents a major unsolved challenge in molecular design. Here, we demonstrate that high-throughput, iterative experimental testing of diverse RNA designs crowdsourced from Eterna yields sensors of increasingly complex functions of input oligonucleotide concentrations. After designing single-input RNA sensors with activation ratios beyond our detection limits, we created logic gates, including challenging XOR and XNOR gates, and sensors that respond to the ratio of two inputs. Finally, we describe the OpenTB challenge, which elicited 85-nucleotide sensors that compute a score for diagnosing active tuberculosis, based on the ratio of products of three gene segments. Building on OpenTB design strategies, we created an algorithm Nucleologic that produces similarly compact sensors for the three-gene score based on RNA and DNA. These results open new avenues for diverse applications of compact, single molecule sensors previously limited by design complexity.

Monitoring of bacterial film formation and its breakdown with an angular-based surface plasmon resonance biosensor.

Bacterial biofilms are a leading cause of infection in health-care settings. Surface plasmon resonance (SPR) biosensors stand as valuable tools not only for the detection of biological entities and the characterisation of biomaterials but also as a suitable means to monitor bacterial film formation. This article reports on a proof-of-concept study for the use of an angular-based SPR biosensor for the monitoring of bacterial cell growth and biofilm formation and removal under the effect of different cleaning agents. The benefit of this custom-made SPR instrument is that it records simultaneously both the critical and resonant angles. This provides unique information on the growth of bacterial cells which is otherwise not obtainable with commonly used intensity-based SPR systems. The results clearly showed that a multilayer biofilm can be formed in 48 hours and the steps involved can be monitored in real-time with the SPR instrument through the measurement of the refractive index change and following the evolution in the shape of the SPR curve. The number, the depth and the sharpness of the reflection ripples varied as the film became thicker. Simulation results confirmed that the number of layers of bacteria affected the number of ripples at the critical angle. Real-time monitoring of the film breakdown with three cleaning agents indicated that bleach solution at 4.5% was the most effective in disrupting the biofilm from the gold sensor. Our overall findings suggest that the SPR biosensor with angular modulation presented in this article can perform real-time monitoring of biofilm formation and has the potential to be used as a platform to test the efficiency of disinfectants.

A nanoparticle-based thermo-dynamic aptasensor for small molecule detection.

Small molecules (MW < 1000 Da) represent a large class of biomarkers of interest. Recently, a new class of biosensors has been emerging thanks to the recognition properties of aptamers, short DNA or RNA single strands, selected against such small molecular targets. Among them, an adenosine-specific aptamer has been largely described and used due to its remarkable affinity to this small target (KD = 6 µM). In this paper, we achieved the proof-of-principle of an aptasensor based on the thermodynamic follow-up of adenosine binding with engineered split-aptamer sequences. The detection is carried out by surface plasmon resonance imaging of split-aptamer micro-arrays, while signal amplification is ensured by gold nanoparticles (AuNPs). This original approach based on DNA sequence engineering and AuNP conjugation enabled us to reach limits of detection (LOD) 200 times lower than the KD measured in solution with the native aptamer (LOD = 30 nM).

Gold Nanoparticles Surface Plasmon Resonance Enhanced Signal for the Detection of Small Molecules on Split-Aptamer Microarrays (Small Molecules Detection from Split-Aptamers).

The detection of small molecules by biosensors remains a challenge for diagnostics in many areas like pharmacology, environment or homeland security. The main difficulty comes from both the low molecular weight and low concentrations of most targets, which generally requires an indirect detection with an amplification or a sandwich procedure. In this study, we combine both strategies as the amplification of Surface Plasmon Resonance imaging (SPRi) signal is obtained by the use of gold nanoparticles and the sequence engineering of split-aptamers, short oligonucleotides strands with strong affinity towards small targets, allows for a sandwich structure. Combining those two strategies, we obtained state-of-the-art results in the limit of detection (LOD = 50 nM) with the model target adenosine. Furthermore, the SPRi detection led on aptamer microarrays paves the way for potential multi-target detections thanks to the multi-probe imaging approach.

Real time monitoring of thrombin interactions with its aptamers: insights into the sandwich complex formation.

Aptamers are raising an increasing interest for biosensor applications as replacements for antibodies due to their high stability and low cost. Thrombin, a key enzyme in the coagulation cascade, is an archetypical target against which two different aptamers, binding to two different exosites, have been selected. Recent studies dedicated to thrombin monitoring applications of biosensors have taken advantage of a potential sandwich-like structure between thrombin and these two aptamers for amplification purposes. However, in most cases, only end-point analysis was observed as a result of labeling requirements, thus preventing access to the kinetics of the complex formation. By using Surface Plasmon Resonance (SPR) imaging of aptamer-functionalized biosensors, we followed the binding of thrombin on the sensor and its interaction with a second reporter aptamer in real-time and in a label-free manner. Surprisingly, we showed that the injection of a second unlabeled-aptamer following the previous thrombin injection destabilized the thrombin-aptamer complex formed on the sensor surface, thus limiting any further amplification. However, the direct co-injection of thrombin, pre-complexed with a biotinylated aptamer bound to streptavidin efficiently increased the SPR signal by comparison to single thrombin detection. The various injection sequences performed may be rationalized considering a poor selectivity of one of the aptamers towards its exosite and a further negative allosteric effect upon sandwich complexation of the thrombin with its aptamers.