一、Introduction

Aptamers are single‑stranded DNA or RNA molecules obtained through in‑vitro SELEX screening. They rely on higher‑order spatial folding to recognize target substances specifically. Conventional magnetic‑bead SELEX needs target immobilization on solid supports. Immobilization modifications may alter native target conformations. Solid‑matrix‑related screening bias appears, and overall screening cycles tend to be long ^[1].

CE‑SELEX was formally established in 2004. It adopts capillary electrophoresis as separation tool. All binding reactions take place in free liquid solution. Conformational disturbances caused by target fixation get avoided. High‑affinity aptamers can be harvested after only a few screening rounds. It represents a representative immobilization‑free SELEX technique ^[2].

二、Technical Principle and Experimental Workflow of CE‑SELEX

Incubate random nucleic‑acid libraries with targets in liquid phase. Certain aptamers fold and bind targets to form molecular complexes. Capillary electrophoresis separates free nucleic acids from target‑aptamer complexes according to differences in charge‑to‑mass ratio. Nucleic‑acid molecules inside collected complexes go through PCR amplification to regenerate libraries. Multiple enrichment cycles finally yield highly specific aptamer sequences ^[3].

The complete experimental workflow contains five steps:

1. Liquid‑phase incubation

Mix nucleic‑acid libraries with targets inside physiologically compatible buffers. Both targets and nucleic‑acid strands maintain native conformations.

2. Capillary electrophoresis separation

Load mixed samples into capillary‑electrophoresis instruments. Apply voltage. Distinct migration behaviours distinguish free nucleic acids from target‑aptamer complexes.

3. Fraction collection

Collect fractions containing target‑bound aptamer complexes precisely according to electropherogram signals. Discard fractions with unbound nucleic‑acid material.

4. Library amplification

Recover nucleic acids from collected fractions. Perform PCR amplification to produce single‑stranded nucleic‑acid libraries for next‑round SELEX screening.

5. Sequencing

Run NGS high‑throughput sequencing once CE‑SELEX cycles finish. Screen promising aptamer candidates.

三、Technical Advantages and Existing Challenges

1.Technical advantages

①1. Target immobilization is unnecessary. All reactions run in free solution. Native target conformations get preserved to the maximum extent ^[1].

2. High screening efficiency. CE‑SELEX usually completes selection within one to four rounds. Aptamer development cycles become notably shorter.

3. High separation resolution from capillary electrophoresis. Minor mobility differences can be told apart. This favours enrichment of high‑affinity aptamer clones.

4. Flexible system settings. Screening buffers can mimic physiological conditions. The technique suits aptamer discovery for protein and other macromolecular targets.

2.Major challenges

①Capillary electrophoresis accepts tiny injection volumes. Total sequence diversity of input libraries becomes limited. Some rare high‑affinity aptamer sequences may get lost.

②This method works better for macromolecular targets such as proteins. Small‑molecule targets generate only subtle mobility shifts after binding. Effective separation hardly takes place, so small‑molecule screening is not suitable.

③Electrophoresis outcomes are sensitive to buffer pH, ionic strength and capillary coating conditions. Considerable parameter‑tuning work is required.

④Accurate adjustment of complex collection windows proves difficult. Improper collection settings directly lead to failed enrichment. Besides, PCR amplification carries inherent risks of amplification bias.

四、Main Application Scenarios of CE‑SELEX

1. Aptamer development for biomarker proteins

CE‑SELEX fits various tumour biomarkers, inflammatory factors and enzyme targets. Benefiting from free‑solution screening, obtained aptamers recognize native protein conformations. They support building fluorescent and electrochemical biosensing platforms for early‑stage in‑vitro‑diagnostic reagent development.

2. Antagonistic‑targeting molecules and lead‑drug development

Aptamers screened via CE‑SELEX can attach to protein active sites. They act as protein antagonists for target‑function inhibition research. They also serve as targeting guiding elements for drug‑delivery‑system research and supply candidate molecular tools for pre‑clinical studies.

3. Combined hybrid‑SELEX screening

CE‑SELEX often serves as primary‑screening tool. It generates a pool of aptamers with good liquid‑phase affinity. Results then feed into Cell‑SELEX for cell‑level selection. This workflow balances liquid‑phase binding performance and cell‑surface‑recognition capacity and offsets limitations from single‑mode screening ^[4].

4. Basic research for molecular interactions

CE‑SELEX screening itself can capture binding‑kinetics data simultaneously. Selected aptamers work as molecular probes for basic biochemical research covering nucleic‑acid‑protein interactions and protein‑conformation studies.

五、CE‑SELEX‑Related Technical Services of KMD‑Bioscience

1. Custom‑tailored experimental schemes

Design overall CE‑SELEX experimental workflows. Optimize reaction‑system parameters and screening round settings. Provide hybrid screening strategies that combine CE‑SELEX with other SELEX techniques for appropriate projects.

2. CE‑SELEX screening

Complete liquid‑phase library incubation, capillary‑electrophoresis separation, precise collection of target‑aptamer complexes, nucleic‑acid recovery and library amplification. Carry out multi‑round SELEX enrichment.

3. High‑throughput sequencing analysis

Perform NGS sequencing for libraries from each enrichment round. Analyse sequence abundance and conserved motifs, and identify aptamer candidates.

4. Activity validation

Synthesize candidate aptamers. Measure liquid‑phase binding affinity and specificity with SPR, MST and other analytical tools.

5. Sample delivery

Hand over complete experimental reports, raw sequencing datasets and synthesized aptamer samples. Offer technical support for diagnostic‑reagent development, drug research and fundamental‑science projects.

FAQs

Q：CE‑SELEX produces enrichment signals after several screening cycles, yet candidate aptamers show very weak affinity toward target proteins. What are the main causes？

1. Improper setting for capillary‑electrophoresis collection windows. Collected fractions contain large amounts of unbound free nucleic‑acid material. Libraries show false enrichment instead of accumulation of genuine aptamer sequences.

2. PCR amplification bias. Certain non‑specific sequences get amplified more efficiently and keep accumulating throughout SELEX cycles.

3. Unreasonable target concentration settings. Excess target concentration enriches many weakly‑binding sequences, which deliver poor affinity in liquid‑phase validation.

4. Large environmental gaps exist between electrophoresis buffers and validation buffers. Aptamer folding conformations get altered and complex‑forming capacity becomes impaired.

参考文献：

[1] Chinchilla-Cárdenas DJ, Cruz-Méndez JS, Petano-Duque JM, et al. Current developments of SELEX technologies and prospects in the aptamer selection with clinical applications. J Genet Eng Biotechnol. 2024;22(3):100400. doi:10.1016/j.jgeb.2024.100400

[2] Zhuo Z, Yu Y, Wang M, et al. Recent Advances in SELEX Technology and Aptamer Applications in Biomedicine. Int J Mol Sci. 2017;18(10):2142. Published 2017 Oct 14. doi:10.3390/ijms18102142

[3]Hu YY, Yang G, Qu F. Se Pu. 2025;43(4):297-308. doi:10.3724/SP.J.1123.2024.04012

[4]Uemachi H, Kasahara Y, Tanaka K, Okuda T, Yoneda Y, Obika S. Hybrid-Type SELEX for the Selection of Artificial Nucleic Acid Aptamers Exhibiting Cell Internalization Activity. Pharmaceutics. 2021;13(6):888. Published 2021 Jun 15. doi:10.3390/pharmaceutics13060888