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Table 1 Advantages and disadvantages of reported common methods for monitoring the aptamer-target binding in SELEX rounds

From: SELEX tool: a novel and convenient gel-based diffusion method for monitoring of aptamer-target binding

Monitoring methodsSuitable targetsAdvantagesLimitationsReferences
Dot blottingProteinFocus 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]
qPCRProtein & Small moleculeRelative ease of performanceFocus on the elution phase, which could be confused by non-specific eluates;
Error of nonspecific amplification;
[35,36,37,38]
EMSAProteinFocus on the candidate aptamers binding phase, which shows the real enrichment in SELEXSequence labeling required;
Labor- and time-consuming;
Not suitable for small molecule targets
[39]
Gel-shiftingProteinFocus 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]
ELONAProteinFocus 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 analysisProtein & Small moleculeRelative ease of performanceFocus on the elution phase, which could be confused by non-specific eluates;
Error of nonspecific amplification
[41]
HTSProtein & Small moleculeFocus on the enrichment of candidate aptamers according to the SELEX roundsExpensive cost required;
Focus on the elution phase, which could confuse by none-specific elution;
Time-consuming for sample preparation
[35, 42]
SPRProtein & Small moleculeFocus on the candidate aptamers binding phase, which shows the real enrichment in SELEXExpensive sensor chip required;
Labor- and time-consuming
[42, 43]
UV quantificationSmall moleculeEase of performanceFocus on the elution phase, which could confuse by none-specific elution[44]
Fluorescence quantificationSmall moleculeRelative ease of performanceSequence labeled with fluorophore required;
Focus on the elution phase, which could confuse by none-specific elution
[45, 46]
Fluorescence binding assaySmall moleculeFocus 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 assaySmall moleculeFocus on the candidate aptamers binding phase, which shows the real enrichment in SELEXTarget-coupled column required;
Labor- and time-consuming
[49]
GBDMProtein & Small moleculeFocus 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 requiredOptimized in This work
  1. 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