Aptamer screening technologies

Date：2025-12-26

In recent years, various screening methods have been developed to generate aptamers more reliably and efficiently. These include Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and its various derivative techniques, such as magnetic bead SELEX, capture SELEX, graphene oxide SELEX, cell-SELEX, capillary electrophoresis SELEX, and atomic force microscopy SELEX. This review summarizes these methods and analyzes the key characteristics, advantages, and limitations of each SELEX approach (see Table 1). The wide application of aptamers has driven the continuous development of SELEX technology. Over recent decades, numerous new methods for screening aptamers have emerged, significantly reducing the screening time from weeks to just hours.

Table 1. Advantages and disadvantages of SELEX method currently used.

Method	Key Aspects	Advantages	Disadvantages
Target small molecule compounds
Agarose affinity chromatography SELEX	The aptamers that can bind to the target can be isolated by fixing the small molecular target with agarose affinity chromatography column	The most traditional aptamer screening method with the longest application time	Low separation efficiency, requires relatively large amounts of elution materials
MB-SELEX	SsDNA or target was fixed with magnetic beads, and the bound sequence was separated from the unbound sequence by magnetic field.	Avoid changes in the inherent structure of the target and improve the affinity of the selected aptamer	Bead aggregations might cause blockage in the micro-channel
Capture-SELEX	Oligonucleotide library is immobilized on a support instead of the targets to identify aptamers against small soluble molecules.	Immobilization of the target not required. Used for the discovery of structure-switching aptamers.	Some oligonucleotides from the library might be not released/selected.
GO-SELEX	Utilized the adsorption of graphene oxide with ssDNA to obtain high affinity aptamers	Immobilization of the target not required. Can be used to screen multi-target aptamers, especially toxic molecules.	Expensive and difficult to apply
Target proteins
CE-SELEX	Involves separation of ions based onelectrophoretic mobility.	Can be performed on non-immobilizedtargets without steric hindrance, the amount of sample and solvent required for capillary electrophoresis are very small. Fast, only few (1–4) rounds of selection required.	Not suitable for small molecules. Expensive equipment.
M-SELEX	Combines SELEX with a microfluidic system.	Rapid. Very efficient (only small amounts of reagents needed). Applicable to small molecules. Automatable.	Low purity/recovery of aptamers. Target immobilization required.
AFM-SELEX	Employs AFM to create three-dimensional image of the sample surface.	Able to isolate high affinity aptamers. Fast (only 3–4 rounds required).	Expensive equipment required. Immobilization of target and aptamers required.
Target whole living cells
Cell-SELEX	Utilizes whole live cells as targets for selection of aptamers.	Prior knowledge of the target not required. Aptamers are selected against molecules in their native state. Many potential targets available on the cell surface. Protein purification not required.	Suitable for cell surface targets. Requires high level of technical expertise. Costly. Time consuming. Post SELEX identification of the target required.

The SELEX Framework and Its Challenges

Classical SELEX involves iterative cycles of selection, partitioning, and amplification from a randomized oligonucleotide library. While effective, the process has historically been labor-intensive, time-consuming (often requiring weeks to months), and prone to nonspecific binding or amplification bias. These limitations spurred the development of numerous SELEX variants, each designed to enhance efficiency, specificity, or applicability to particular target types.

Key SELEX Modifications and Their Applications

Magnetic Bead SELEX (MB-SELEX)
By immobilizing targets on magnetic beads, this approach simplifies the separation of bound and unbound sequences. It reduces handling time and is particularly effective for protein targets, though it may be influenced by bead surface chemistry.
Cell-SELEX
This method uses whole living cells as targets, enabling the selection of aptamers against native membrane proteins or cellular epitopes without prior purification. It is invaluable for cancer biomarker discovery but may yield aptamers with uncertain molecular targets.
Capillary Electrophoresis SELEX (CE-SELEX)
Leveraging the high resolution of capillary electrophoresis, CE-SELEX separates target-bound sequences based on mobility shifts. It significantly shortens selection cycles (sometimes to a single round) and improves specificity, though it requires specialized instrumentation.
Capture-SELEX
Particularly useful for small molecules, this technique immobilizes the oligonucleotide library rather than the target. It facilitates selection against soluble, non-immobilizable targets and supports aptamer discovery for metabolites or toxins.
Graphene Oxide SELEX (GO-SELEX)
Graphene oxide adsorbs single-stranded DNA, quenching fluorescence of unbound sequences. It offers a label-free, rapid screening option, often integrated with fluorescence detection for real-time monitoring.
Atomic Force Microscopy SELEX (AFM-SELEX)
A less common but emerging approach, AFM-SELEX uses nanomechanical sensing to detect binding events at the single-molecule level. It holds promise for ultrasensitive selections but remains technically demanding.

Technological Convergence and Accelerated Screening

Recent innovations have progressively reduced selection time from weeks to hours. Microfluidic SELEX platforms, for instance, enable automation and minute reagent consumption, while high-throughput sequencing allows deep analysis of enriched libraries. Machine learning algorithms are now being incorporated to predict aptamer-target interactions, guiding library design and shortening experimental cycles.

Future Directions

The convergence of SELEX with nanotechnology, synthetic biology, and computational modeling is paving the way for next-generation aptamer discovery. Emerging methods focus on in silico screening, multi-target parallel selection, and functional aptamer integration into biosensors or therapeutic delivery systems.

Conclusion

The landscape of aptamer screening has evolved from a single, cumbersome process into a diverse toolkit of precision methods. As SELEX variants become more integrated, automated, and intelligent, the pace of aptamer development will continue to accelerate, unlocking new opportunities in personalized medicine, environmental monitoring, and molecular diagnostics.

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