Table 1.
Advantages and disadvantages of reported common methods for monitoring the aptamer-target binding in SELEX rounds
| Monitoring methods | Suitable targets | Advantages | Limitations | References |
|---|---|---|---|---|
| Dot blotting | Protein |
Focus on the candidate aptamers binding phase, which shows the real enrichment in SELEX; Relative ease of performance |
Sequence labeling required; Not suitable for the target molecules with the same electrostatic charge as NC membrane used |
[34] |
| qPCR | Protein & Small molecule | Relative ease of performance |
Focus on the elution phase, which could be confused by non-specific eluates; Error of nonspecific amplification; |
[35–38] |
| EMSA | Protein | Focus on the candidate aptamers binding phase, which shows the real enrichment in SELEX |
Sequence labeling required; Labor- and time-consuming; Not suitable for small molecule targets |
[39] |
| Gel-shifting | Protein |
Focus on the candidate aptamers binding phase, which shows the real enrichment in SELEX; Easy for performance |
Detection in none-binding conditions, I.E. electrophoretic buffer solution; Not suitable for small molecule targets |
[34, 36] |
| ELONA | Protein |
Focus on the candidate aptamers binding phase, which shows the real enrichment in SELEX; Relative ease of performance |
Sequence labeling required; Labor- and time-consuming; Non-specific binding of candidates to the plate, which confuse the enrichment; Not suitable for small molecule targets |
[40] |
| Agarose gel analysis | Protein & Small molecule | Relative ease of performance |
Focus on the elution phase, which could be confused by non-specific eluates; Error of nonspecific amplification |
[41] |
| HTS | Protein & Small molecule | Focus on the enrichment of candidate aptamers according to the SELEX rounds |
Expensive cost required; Focus on the elution phase, which could confuse by none-specific elution; Time-consuming for sample preparation |
[35, 42] |
| SPR | Protein & Small molecule | Focus on the candidate aptamers binding phase, which shows the real enrichment in SELEX |
Expensive sensor chip required; Labor- and time-consuming |
[42, 43] |
| UV quantification | Small molecule | Ease of performance | Focus on the elution phase, which could confuse by none-specific elution | [44] |
| Fluorescence quantification | Small molecule | Relative ease of performance |
Sequence labeled with fluorophore required; Focus on the elution phase, which could confuse by none-specific elution |
[45, 46] |
| Fluorescence binding assay | Small molecule |
Focus on the candidate aptamers binding phase, which shows the real enrichment in SELEX; Relative ease of performance |
The autofluorescence of target required | [47, 48] |
| Gel-elution assay | Small molecule | Focus on the candidate aptamers binding phase, which shows the real enrichment in SELEX |
Target-coupled column required; Labor- and time-consuming |
[49] |
| GBDM | Protein & Small molecule |
Focus on the candidate-target binding phase, which shows the real enrichment in SELEX; Easy for performance without expensive equipment; Suitable for monitoring every selection step during SELEX process |
Overnight diffusion required | Optimized in This work |
Notes: qPCR real-time quantitative PCR, EMSA Electrophoretic mobility shift assay, ELONA enzyme-linked oligonucleotide assay, HTS High throughput sequencing, SPR Surface plasmon resonance, GBDM gel-based diffusion method