Results

Stage II Summary
Design and Testing of Molecular Biology Protocols for a Nanostructured Platform with Isothermal Amplification for Rapid DNA Detection
Point-of-care multiplex biosensors (xPOC) enable the simultaneous detection of multiple analytes from a single sample in a timely manner, leading to immediate clinical decision-making. In this context, isothermal amplification (IA) methods are suitable for the genomic identification of infectious agents. Microarray systems can be adopted in xPOC as they allow parallel analysis of biomolecules.
This project aims to address the urgent need for xPOC systems for detecting various pathogens (bacteria/fungi). Therefore, the main objective is to develop a nanostructured detection platform implementing an IA method for rapid DNA amplification. This issue has been insufficiently addressed in Romanian research.
The project focuses on the following aspects:

• Developing a nanostructured Si platform with a functionalized surface suitable for binding chemically modified probes.

• Manufacturing PDMS channels serving as reaction chambers for IA.

• Designing primer sets and optimizing IA reactions.

• Performing statistical data analysis to demonstrate the specificity of the xPOC platform.

The overall goal of the project is to develop a nanostructured detection platform implementing solid-phase isothermal amplification for the rapid detection of DNA specific to bacteria/yeast.
The specific activities for Stage 2 were:
A. 2.1. Optimization of molecular biology protocols in isothermal amplification.
A. 2.2. Design, manufacturing, and preliminary testing of the biosensor.
A. 2.3. Validation of isothermal amplification through the PCR method.

The specific activities of designing, manufacturing, and testing the biosensor have been completed. Consequently, the collection of all reference strains of bacteria and yeast has been finalized. Among all primer sets, those that showed no cross-reactivity and were validated in both PCR and classic RPA reactions are presented. Active areas were defined through 1-MACE chemical etching, and the resulting structures were encapsulated in PDMS. The RPA reaction was tested on the obtained structures, and the results were promising.

______________

Estimated results

SPIonNANODET project will raise the technical-scientific and innovation standards of the host institute by harnessing the following result indicators, as follows:
- Sample collection
- 1 method for primer design
- 1 protocol for DNA labelling
- 1 optimized method for obtaining a nanostructured silicon substrate
- 1 optimized functionalization method
- 1 optimized method for primer dispensing and immobilization on biochip
- 1 optimized protocol for PDMS microwell fabrication
- 1 protocol for solid-phase amplification
- At least 30 biochips manufactured and tested
- 1 optimised statistical analysis flow using R language
- At least 10 PCR tests for confirming the solid-phase isothermal amplification on the developed biochips
- Dissemination of results by attending 2 international conferences
- 2 ISI papers