Plastic surface-immobilized primers for integrated DNA capture and purification in simplified molecular diagnostics - a proof-of-concept study

Introduction Current molecular diagnostic (MDx) platforms rely on silica-coated surfaces for nucleic acid extraction. This requires multiple wash buffers and complex automation, thereby creating cost and usability barriers for resource-limited settings. Several modifications of MDx towards affordability adopt miniaturization technologies such as microfluidics, underscoring the potential impact of material reduction on MDx cost. Most advancements, however, replicate the same silica-based process steps leaving its cost-implication unaddressed. This study positions oligonucleotide-functionalized surfaces as a potential alternative approach for DNA capture with the potential for future integration into lab-on-a-chip (LoC) technologies. Methods Hybridization-based genomic DNA and oligo capture were first validated using oligonucleotide-functionalized microparticles as a surrogate for polycarbonate plastic surfaces. Polycarbonate plastic was selected due to its biocompatibility, thermal tolerance, and the possibility of primer immobilization through amide bond. To enable the immobilization of a high concentration of oligonucleotides on polycarbonate plastics, their tubes were sequentially treated through acetone splash (10 s exposure) and UV photo-oxidation (10 min) to increase their carboxyl density. Results Combined acetone and UV treatments increased the tubes’ carboxyl density 2- fold (2.02 × 10 –3 to 4.0 × 10 –3 nmol/mm 2 ). To further improve the primer-carrying capacity of the tube surface, the oligonucleotide loading capacity of two linker molecules, branched polyethyleneimine (BPEI) and ethylenediamine (EA), was compared. BPEI linkers provided 2.6-fold higher oligonucleotide payload compared to ethylenediamine. The concentration of surface-immobilized oligonucleotides available for DNA capture was quantified fluorometrically from RFU values referenced against a calibration curve. An average of 0.125 ± 0.018 μ M and 0.0296 ± 0.0132 μ M oligonucleotides were immobilized in a 25 μ L working volume using BPEI and EA respectively. Discussion Although the concentration achieved using BPEI is theoretically compatible with 2-primer amplification chemistries such as PCR and recombinase polymerase amplification (RPA), further optimization is required to enable LAMP. Optimization efforts addressing the tubes’ tapered geometry and the need for mixing during functionalization is recommended to achieve higher primer concentrations. This proof-of-concept establishes preliminary technical feasibility of primer-functionalized surfaces as a potential alternative to silica-based nucleic acid extraction, pending full workflow integration. The validated surface engineering approach provides a development pathway for a cost-effective diagnostic platforms suitable for resource-limited settings.