Rikenella microfusus is an essential intestinal probiotic with great potential. The latest research shows that imbalance in the intestinal flora are related to the occurrence of various diseases, such as intestinal diseases, immune diseases, and metabolic diseases. Rikenella may be a target or biomarker for some diseases, providing a new possibility for preventing and treating these diseases by monitoring and optimizing the abundance of Rikenella in the intestine. However, the current monitoring methods have disadvantages, such as long detection times, complicated operations, and high costs, which seriously limit the possibility of clinical application of microbiome-based treatment options. Therefore, the intention of this study was to evolve an enriched aptamer library to be used for specific labeling of R. microfusus, allowing rapid and low-cost detection methods and, ultimately the construction of aptamer-based biosensors. In this study, we used Rikenella as the target bacterium for an in vitro whole Cell-SELEX (Systematic Evolution of Ligands by EXponential Enrichment) to evolve and enrich specific DNA oligonucleotide aptamers. Five other prominent anaerobic gut bacteria were included in this process for counterselection and served as control cells. The aptamer library R.m-R13 was evolved with high specificity and strong affinity (K_d = 9.597 nM after 13 rounds of selection). With this enriched aptamer library, R. microfusus could efficiently be discriminated from the control bacteria in complex mixtures using different analysis techniques, including fluorescence microscopy or fluorometric suspension assays, and even in human stool samples. These preliminary results open new avenues toward the development of aptamer-based microbiome bio-sensing applications for fast and reliable monitoring of R. microfusus.

Roseburia intestinalis has received attention as a potential probiotic bacterium. Recent studies have demonstrated that changes in its intestinal abundance can cause various diseases, such as obesity, enteritis and atherosclerosis. Probiotic administration or fecal transplantation alter the structure of the intestinal flora, offering possibilities for the prevention and treatment of these diseases. However, current monitoring methods, such as 16S rRNA sequencing, are complex and costly and require specialized personnel to perform the tests, making it difficult to continuously monitor patients during treatment. Hence, the rapid and cost-effective quantification of intestinal bacteria has become an urgent problem to be solved. Aptamers are of emerging interest because their stability, low immunogenicity and ease of modification are attractive properties for a variety of applications. We report a FluCell-SELEX polyclonal aptamer library specific for R. intestinalis isolated after seven evolution rounds, that can bind and label this organism for fluorescence microscopy and binding assays. Moreover, R. intestinalis can be distinguished from other major intestinal bacteria in complex defined mixtures and in human stool samples. We believe that this preliminary evidence opens new avenues towards aptamer-based electronic biosensors as new powerful and inexpensive diagnostic tools for the relative quantitative monitoring of R. intestinalis in gut microbiomes.

Recent studies have demonstrated that changes in the abundance of the intestinal bacterium Blautia producta, a potential probiotic, are closely associated with the development of various diseases such as obesity, diabetes, some neurodegenerative diseases, and certain cancers. However, there is still a lack of an effective method to detect the abundance of B. producta in the gut rapidly. Especially, DNA aptamers are now widely used as biometric components for medical testing due to their unique characteristics, including high chemical stability, low production cost, ease of chemical modification, low immunogenicity, and fast reproducibility. We successfully obtained a high-affinity nucleic acid aptamer library (B.p-R14) after 14 SELEX rounds, which efficiently discriminates B. producta in different analysis techniques including fluorometric suspension assays or fluorescence microscopy from other major gut bacteria in complex mixtures and even in human stool samples. These preliminary findings will be the basis towards aptamer-based biosensing applications for the fast and reliable monitoring of B. producta in the human gut microbiome.

Here we present for the first time a potential wound dressing material implementing aptamers as binding entities to remove pathogenic cells from newly contaminated surfaces of wound matrix-mimicking collagen gels. The model pathogen in this study was the Gram-negative opportunistic bacterium Pseudomonas aeruginosa, which represents a considerable health threat in hospital environments as a cause of severe infections of burn or post-surgery wounds. A two-layered hydrogel composite material was constructed based on an established eight-membered focused anti-P. aeruginosa polyclonal aptamer library, which was chemically crosslinked to the material surface to form a trapping zone for efficient binding of the pathogen. A drug-loaded zone of the composite released the C14R antimicrobial peptide to deliver it directly to the bound pathogenic cells. We demonstrate that this material combining aptamer-mediated affinity and peptide-dependent pathogen eradication can quantitatively remove bacterial cells from the “wound” surface, and we show that the surface-trapped bacteria are completely killed. The drug delivery function of the composite thus represents an extra safeguarding property and thus probably one of the most important additional advances of a next-generation or smart wound dressing ensuring the complete removal and/or eradication of the pathogen of a freshly infected wound.

Single-stranded DNA aptamers as affinity molecules for the rapid, reliable detection of intestinal bacteria are of particular interest to equip health systems with novel robust and cheap diagnostic tools for monitoring the success of supplementation strategies with selected probiotic gut bacteria in the fight against major widespread threats, such as obesity and neurodegenerative diseases. The human gut bacterium Parabacteroides distasonis (P. distasonis) is positively associated with diseases such as obesity, non-alcoholic fatty liver disease and multiple sclerosis with reduced cell counts in these diseases and is thus a promising potential probiotic bacterium for future microbial supplementation. In this paper we report on the evolution of a specific polyclonal aptamer library by the fluorescence based FluCell-SELEX directed against whole cells of P. distasonis that specifically and efficiently binds and labels P. distasonis. The aptamer library showed high binding affinity and was suited to quantitatively discriminate P. distasonis from other prominent gut bacteria also in mixtures. We believe that this library against a promising probiotic bacterium as a prototype may open new routes towards the development of novel biosensors for the easy and efficient quantitative monitoring of microbial abundance in human microbiomes in general.