Electrochemical monitoring of enzymatic cleavage in nanochannels with nanoparticle-based enhancement: determination of MMP-9 biomarker

  1. A unique way of detecting molecules
  2. A groundbreaking research around MMP-9 biomarker
  3. Abyntek’s role
  4. Conclusion

Sensors based on nanochannels consist of the alteration of the ionic flow through a nanopore by the presence of unique analytes that cross it under the application of an external potential. This principle has been applied to the detection of single molecules such as DNA, RNA, Peptides or proteins or in this case, the MMP-9 biomarker.

A unique way of detecting molecules

Although the first nanopores used as sensors were of biological origin, solid nanoporous membranes have emerged as a preferable alternative, as they facilitate the detection of larger molecules.

The use of this method to detect smaller particles is largely unexplored, since they do not produce significant enough clogging of the nanopores for accurate measurements.

However, for the first time in history, the use of nanoparticles as carriers of an enzymatic substrate immobilised within nanoporous aluminium membranes is proposed to amplify the blocking of the nanochannel and, therefore, improving the detection capacity of an enzyme.

A groundbreaking research around MMP-9 biomarker

Streptavidin-modified polystyrene particles (PSNPs) were proposed as carrier agents, applied to the detection of a biomarker that is overexpressed in different types of cancer, neurodegenerative diseases and chronic wounds: metalloproteinase 9 (MMP-9).

MPP-9 is a gelatinase enzyme that has specific reactivity on the peptide Leu-Gly-Arg-Met-Gly-Leu-Pro-Gly-Lys (PEP), so this was used in conjunction with PSNP to detect MMP. -9.

The incorporation of nanoparticles amplifies the blocking produced both sterically and electrostatically by introducing a high charge density into the channel. These charges depend on the pH of the measurement buffer, so this parameter was studied in detail in this research.

Furthermore, the interaction with the charges generated inside the nanochannel, positively ([Ru(NH3)6]3+) ​​and negatively ([Fe(CN)6]4−) charged redox indicators, were evaluated for the voltammetric measurements to determine the blockade obtained, making this study a pioneer in the relevance of this class of detection systems, whose conclusions can be extended to other analytes and biomarkers.

Abyntek’s role

The peptide Leu-Gly-Arg-Met-Gly-Leu-Pro-Gly-Lys (PEP) biotinylated in N-Temrinal was provided by Abyntek Biopharma.

Abyntek Biopharma has more than 15 years dedicated to providing the best possible service to researchers. That is why we put all our know-how and experience into helping thousands of scientists find the perfect reagent for their research. And why we were able to bring together all the experience of our scientific department to help researchers like the participants in this study through our custom reagent synthesis service.

In this case, the researchers turned to Abyntek’s custom peptide synthesis service. Using state-of-the-art bioinformatics applications, as well as the experience of our scientific team, the peptide was synthesized using a solid phase synthesis process.

After the synthesis of the peptide, the quality of the peptide was corroborated by chromatography and mass spectrometry, as well as a solubility test before delivering it directly to the researchers, who were able to use it to publish their discoveries in a first-quartile journal (Q1).

In conclusion

Under optimal conditions, metalloprotein 9 (MMP-9) is detected at clinically relevant levels (between 100 and 1200 ng/mL) with a limit of detection of 75 ng/mL and a limit of quantification of 251 ng/mL, showing a good reproducibility and selectivity.

In addition, it has demonstrated excellent results in real samples with recovery percentages between 80% and 110%.

The approach used by the researchers represents a rapid and inexpensive screening methodology with great potential for diagnosis directly at the medical centre, and continued exploration of these techniques will undoubtedly demonstrate the many applications of that phenomenon.

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