Drop-by-Drop Addition of Reagents to a Double Emulsion.

Developments in droplet microfluidics have facilitated an era of high-throughput, sensitive single-cell, or single-molecule measurements capable of tackling the heterogeneity present in biological systems. Relying on single emulsion (SE) compartments, droplet assays achieve absolute quantification of nucleic acids, massively parallel single-cell profiling, and more. Double emulsions (DEs) have seen recent interest for their potential to build upon SE techniques. DEs are compatible with flow cytometry enabling high-throughput multi-parameter drop screening and eliminate content mixing due to coalescence during lengthy workflows. Despite these strengths, DEs lack important technical functions that exist in SEs such as methods for adding reagents to droplets on demand. Consequently, DEs cannot be used for multistep workflows which has limited their adoption in assay development. Here, strategies to enable reagent addition and other active manipulations on DEs are reported by converting DE inputs to SEs on chip. After conversion, drops are manipulated using existing SE techniques, including reagent addition, before reforming a DE at the outlet. Device designs and operation conditions achieving drop-by-drop reagent addition to DEs are identified and used as part of a multi-step aptamer screening assay performed entirely in DE drops. This work enables the further development of multistep DE droplet assays.

Aptamer Screening: Current Methods and Future Trend towards Non-SELEX Approach.

Aptamers are nucleic acid sequences that specifically bind with target molecules and are vital to applications such as biosensing, drug development, disease diagnostics, etc. The traditional selection procedure of aptamers is based on the Systematic Evolution of Ligands by an Exponential Enrichment (SELEX) process, which relies on repeating cycles of screening and amplification. With the rapid development of aptamer applications, RNA and XNA aptamers draw more attention than before. But their selection is troublesome due to the necessary reverse transcription and transcription process (RNA) or low efficiency and accuracy of enzymes for amplification (XNA). In light of this, we review the recent advances in aptamer selection methods and give an outlook on future development in a non-SELEX approach, which simplifies the procedure and reduces the experimental costs. We first provide an overview of the traditional SELEX methods mostly designed for screening DNA aptamers to introduce the common tools and methods. Then a section on the current screening methods for RNA and XNA is prepared to demonstrate the efforts put into screening these aptamers and the current difficulties. We further predict that the future trend of aptamer selection lies in non-SELEX methods that do not require nucleic acid amplification. We divide non-SELEX methods into an immobilized format and non-immobilized format and discuss how high-resolution partitioning methods could facilitate the further improvement of selection efficiency and accuracy.

Viral aptamer screening and aptamer-based biosensors for virus detection: A review.

Virus-induced infectious diseases have a detrimental effect on public health and exert significant influence on the global economy. Therefore, the rapid and accurate detection of viruses is crucial for effectively preventing and diagnosing infections. Aptamer-based detection technologies have attracted researchers' attention as promising solutions. Aptamers, small single-stranded DNA or RNA screened via systematic evolution of ligands by exponential enrichment (SELEX), possess a high affinity towards their target molecules. Numerous aptamers targeting viral marker proteins or virions have been developed and widely employed in aptamer-based biosensors (aptasensor) for virus detection. This review introduces SELEX schemes for screening aptamers and discusses distinctive SELEX strategies designed explicitly for viral targets. Furthermore, recent advances in aptamer-based biosensing methods for detecting common viruses using different virus-specific aptamers are summarized. Finally, limitations and prospects associated with developing of aptamer-based biosensors are discussed.

A Novel in silico SELEX Method to Screen and Identify Aptamers against Vibrio cholerae.

BACKGROUND: Vibrio cholerae, the causative agent of cholera, has been responsible for global epidemics and many other problems over the centuries. It is one of the main public health issues in less-developed and developing countries and is considered one of the deadliest infectious agents. Therefore, precise and susceptible detection of V. cholerae from environmental and biological samples is critical. Aptamers provide a rapid, sensitive, highly specific, and inexpensive alternative to traditional methods. OBJECTIVE: The present study develops a new protocol inspired by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to implement an in silico aptamer selection against V. cholerae, which can also be employed in the case of other pathogenic microorganisms. METHODS: First, we built an oligonucleotide pool and screened it based on the secondary structure. Following that, we modeled the tertiary structures of filtered sequences and performed RNAprotein dockings to assess binding affinities between RNA sequences and Outer Membrane Protein U (OmpU), an effective marker in distinguishing epidemic strains of V. cholerae, which constitute up to 60% of the total outer membrane protein. Finally, we used molecular dynamics simulation to validate the results. RESULTS: Three sequences (ChOmpUapta) were proposed as final aptameric candidates. Analysis of the top-ranked docking results revealed that these candidate aptamers bound to all subunits of OmpU at the extracellular side with high affinity. Moreover, ChOmpUapta-3 and ChOmpUapta-2 were fully stable and formed strong bonds under dynamic conditions. CONCLUSION: We propose incorporating these candidate sequences into aptasensors for V. cholerae detection.

Ricin aptamer screening based on the QSAR model and construction of piezoresistive micro-cantilever aptasensor.

Currently, many aptamers of different target molecules have been screened by systematic evolution of ligands by exponential enrichment (SELEX) method. However, since the crystal structure of aptamers is complicated to obtain, few studies focus on the quantitative structure activity relationship (QSAR) of aptamers. It is significant to develop a virtual screening method for aptamers based on the QSAR model. In this study, a piezoresistive micro-cantilever aptasensor was fabricated to quantificationally detect ricin based on a new aptamer obtained via a virtual screening method. The advanced screening method based on the established QSAR model can screen aptamers formed by site-directed mutation of bases. The results of intermolecular interactions and molecular docking displayed that the new aptamer was mainly bound to ricin A-chain, and its combination of ricin with ricin B-chain aptamer has a significant synergistic effect. Due to the aptamer with excellent performance and the good linear relationship between the relative resistance change and the vertical deformation of the micro-cantilever, the piezoresistive micro-cantilever aptasensor emerged a linear detection range from 7.5 pg mL-1 to 75 ng mL-1 (R2 = 0.949) with the limit of detection 750 fg mL-1 (S/N ≥ 3) for ricin. Furthermore, the proposed piezoresistive micro-cantilever sensor displayed excellent analytical performance with good reproducibility and high specificity.

A New Design for the Fixed Primer Regions in an Oligonucleotide Library for SELEX Aptamer Screening.

Single-stranded DNA or RNA oligonucleotides, often called aptamers, can bind to a molecular target with both high affinity and selectivity due to their distinct three-dimensional structures. A technique called systematic evolution of ligands by exponential enrichment (SELEX) is used to screen aptamers from a random DNA or RNA pool, or library. The traditionally-designed oligonucleotides in libraries contain a randomized central region along with a fixed primer region at each end for amplifying target-bound central sequences. The single-stranded forward and reverse primer sequences may interfere with target-binding to the central region, resulting in a partial or complete loss of high-affinity aptamers during the SELEX process. To address this issue, researchers have modified the traditional oligonucleotide libraries and developed new types of oligonucleotide libraries; however, these approaches come with various limitations. The author proposes a new design that uses a conformation-changeable sequence as primers, which may open a new avenue for developing an optimized aptamer sequence with both high affinity for, and selective binding to, a particular target via SELEX.