CE-SELEX (Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment) is an innovative technology platform that combines the high-resolution separation capability of capillary electrophoresis with the classic SELEX selection strategy. Traditional SELEX relies on solid‑phase carriers (e.g., agarose beads, magnetic beads) to immobilize the target and enriches high‑affinity aptamers through multiple rounds of “binding‑washing‑elution‑amplification” cycles. This typically requires 8-16 or even more rounds, takes 2-6 months, involves cumbersome operations per round, and suffers from issues such as high non‑specific adsorption and a lack of transparency during the selection process.

In contrast, the core advantages of CE‑SELEX lie in its homogeneous screening format and ultra‑high separation efficiency. The technology does not require target immobilization; target and nucleic acid libraries are incubated directly in free solution. Capillary electrophoresis then physically separates the “target‑nucleic acid complex” from “free nucleic acid” based on differences in electrophoretic mobility under an electric field. Because of the extremely high separation efficiency of capillary electrophoresis (theoretical plate numbers can reach hundreds of thousands to millions), high‑affinity candidate sequences are typically obtained after only 1-4 rounds of selection, compressing the entire screening cycle to several weeks up to two months. In addition, CE‑SELEX offers advantages such as real‑time monitoring of the selection process, low sample consumption (nanoliter‑scale injection), and a high degree of automation, making it particularly suitable for screening target proteins that are sensitive to their native conformation.

The screening cycle is a core metric for evaluating the efficiency of aptamer selection technologies. Traditional SELEX relies on multiple rounds of “binding‑washing‑elution‑amplification” cycles, and due to limited removal efficiency of non-specific sequences in each round, it typically requires 8-16 or more rounds of repeated selection, with the entire cycle taking 2-6 months. Such long timelines not only increase time costs but may also lead to the loss of high‑affinity sequences due to PCR amplification bias.

CE‑SELEX fundamentally changes this situation by leveraging the ultra-high separation efficiency of capillary electrophoresis. Studies have shown that using capillary electrophoresis for separation and selection, an enriched library that specifically recognizes the target can be obtained in as few as 4 rounds. Other reports indicate that CE‑SELEX can reduce the number of selection rounds to 1-3. Overall, CE‑SELEX typically compresses the screening cycle to one‑third to one‑sixth of that of traditional SELEX, i.e., from months down to weeks (approximately 4-8 weeks). This efficiency gain is mainly attributable to the following: capillary electrophoresis can achieve high‑resolution separation of target‑nucleic acid complexes from free nucleic acid within minutes, with separation performance far superior to traditional membrane filtration or magnetic bead washing methods. Moreover, CE‑SELEX allows the collection of more high‑affinity candidate sequences in each round, reducing the impact of amplification bias.

CE-SELEX technology has broad target applicability, covering proteins, peptides, small molecules, cells, and even microplastics. Compared to traditional SELEX, CE‑SELEX offers unique advantages for the following target categories:

Protein targets are the most mature application area for CE‑SELEX. Because CE-SELEX is performed in free solution, it does not require immobilization of the protein onto a solid‑phase carrier. This preserves the native conformation and accessibility of active sites to the greatest extent, avoiding conformational changes or site masking caused by immobilization. This is particularly important for membrane proteins, conformation‑sensitive enzymes, or signaling molecules.

Small-molecule targets have traditionally been challenging for aptamer selection due to their low molecular weight and limited number of available binding groups for nucleic acids. Conventional immobilization methods tend to introduce steric hindrance or alter the conformation of small molecules. As a representative of "target-free" selection technologies, CE‑SELEX enables homogeneous interaction between small molecules and nucleic acid libraries in free solution, avoiding false positives caused by immobilization. Studies have shown that CE-SELEX typically requires fewer rounds to select small‑molecule aptamers, and the obtained aptamers exhibit higher affinity (down to the nmol/L level).

Cell targets:
CE‑SELEX can also be used for aptamer selection against intact live cells. However, it should be noted that cell migration behavior in the capillary is relatively complex, and electrophoretic conditions typically need to be optimized to reduce cell sedimentation and aggregation. Alpha Lifetech has experience with CE‑SELEX selection for diverse target types (proteins, small molecules, cells, peptides) and can customize the most appropriate screening strategy based on the client's target characteristics.

For difficult‑to‑express or conformation‑sensitive target proteins (e.g., membrane proteins, transcription factors, kinases), traditional SELEX faces two major challenges: (1) obtaining sufficient quantities of high‑purity, native‑conformation protein, and (2) the immobilization process may disrupt the active conformation of the protein, leading to selection failure. Alpha Lifetech has developed strategies to improve the success rate:

1) Multi‑host expression systems and optimization strategies:
Alpha Lifetech offers four major expression platforms: prokaryotic, yeast, insect, and mammalian. Personalized solutions are developed for different types of difficult‑to‑express proteins. For example, for membrane proteins requiring glycosylation, mammalian (HEK293/CHO) or insect cell expression systems are recommended. For proteins rich in disulfide bonds, oxidative hosts or co-expression with auxiliary folding factors can be selected. Strategies such as codon optimization, fusion tag selection (His, GST, Fc, etc.), and expression condition optimization (temperature, inducer concentration, duration) are used to maximize soluble expression yield.

2) Non‑immobilized CE‑SELEX homogeneous selection:
One of the greatest advantages of CE-SELEX is that it does not require target immobilization; incubation and separation of the nucleic acid library with the target are performed directly in free solution. This fundamentally avoids the potential impact of immobilization on protein conformation and is especially suitable for conformation‑sensitive proteins, those with poor thermal stability, or those difficult to conjugate to solid‑phase carriers. Clients can provide the protein sample in its native state, and Alpha Lifetech performs the entire selection process in a buffer system approximating physiological conditions.

3) Quality control and activity validation:
Each batch of expressed target protein undergoes SDS‑PAGE, Western Blot, and activity validation (e.g., enzymatic activity, binding activity) to ensure that the protein delivered to the selection step possesses the correct spatial conformation and biological function.