A rapid ELISA method for the detection of sarcosine using pseudoperoxidase activity of gold nanoparticles
Abstrakt
Sarcosine is an amino acid that is formed by methylation of glycine and is present in trace amounts in the body. Increased sarcosine concentrations in blood plasma and urine are associated with sarcosinemia and some other diseases such as prostate cancer.
In addition, sarcosine may be administered as a drug in some psychiatric disorders. Primarily, sarcosine in biological fluids is analyzed using liquid chromatography techniques, but this technique is not suitable for routine analysis.
For this purpose, sarcosine detection using the nanomedicine approach was proposed. In the presented test, Au20 gold nanoparticles (size 20-30 nm, zeta potential -38 mV, absorption maximum at 529 nm), which exhibit pseudoperoxidase activity and convert a suitable substrate such as TMB, ABTS (1 mM) to a distinctive color product, were used.
In the following experiments, the Au20 nanoparticle was linked to the chicken anti-sarcosine antibody. Subsequently, the ELISA method for the determination of sarcosine was prepared.
Firstly, the construct for direct detection of sarcosine bound to the antibody was prepared by both the direct and the indirect method (dependencies were strictly linear, R-2 = 0.996). The subsequent construct uses rabbit antibodies against the anti-sarcosine chicken antibody labelled with Au20 nanoparticle.
The bound product is then monitored by the conversion of the TMB (655 nm) or ABTS (425 nm) substrate (the dependencies are strictly linear, R-2 = 0.998). To further increase the sensitivity of the method (for more than 90% of the original signal), an aggregation agent was added to the used Au20 nanoparticles, which further increased adherence of gold nanoparticles to the antibodies.
This procedure succeeded in achieving subnanomolar detection limits for the amino acid sarcosine.