week0
Protein plays a significant role in performing physiological functions[1], however, in diseased cells, protein carrying out a certain function may indicate the proceedings of disease. Such protein could be sorted to biomarkers, which have been regarded as the targets of disease detection and treatment in recent years.
week1
[2]~[4] Therefore, detecting those biomarkers of protein-type becomes more and more critical to biological and medical fields. There are two main detecting approaches to detect a particular protein in a complex sample. One is direct determination of the content after purification, another is binding assays which include a target recognition probe and a signal transducer.[6]~[9] They are commonly selected from random sequence libraries, using the systematic evolution of ligands by exponential enrichment (SELEX) techniques.[10] Advantages of aptamers over antibodies include longer shelf life, improved thermal stability and ease of modification and conjugation.[11] An interesting binding assay is to use aptamers as the target recognition probes and CRISPR-Cas12a(Cpf1) as the signal amplifier, which is called Aptamer Based Cell-free Detection system(ABCD system, Figure 1). We developed this system to detect those biomarkers of protein-type for the purpose of disease detection or staging.
week2
The former approach includes gel filtration chromatography, ion exchange chromatography and nickel column and more, but these methods have high costs, strict equipment requirements and other issues, which are not suitable for promotion and application. The enzyme-linked immunosorbent assay (ELISA) is a typical representative of the latter approach, nevertheless, such assays using antibodies as affinity ligands have cross-reactivity of antibodies compromising the specificity to the target of interest.
week3
[5] What’s worse, the premise of using ELISA is the need to find the corresponding antibodies, but the fact is that not all proteins can find their specific antibody protein, so the use of ELISA is also limited. Binding assays using aptamers as affinity ligands to recognize specific proteins are better than those using antibodies. Aptamers are short, synthetic single stranded oligonucleotides (DNA or RNA) that can bind to target molecules with high affinity and specificity.
