Rapid on-chip nucleic acid extraction for point-of-care salivary diagnostics

Area of Science:

• Molecular Diagnostics
• Biotechnology
• Point-of-Care Technologies

Background:

• Traditional nucleic acid extraction is complex and unsuitable for point-of-care (POC) settings.
• Efficient RNA extraction from saliva at POC is challenging due to degradation.
• Existing methods require laborious, costly laboratory procedures.

Purpose of the Study:

• To develop a simplified, cost-effective on-chip nucleic acid extraction method for saliva.
• To optimize conditions for RNase inactivation, viral lysis, nucleic acid binding, and elution.
• To demonstrate the compatibility of the method with downstream molecular diagnostic applications.

Main Methods:

• Formulation of a custom buffer (POC-Pure) for silica-based nucleic acid binding and elution.
• Fabrication of a microfluidic chip using xurography and laser cutting with a three-way valve.
• Optimization of RNase inactivation, viral lysis, and nucleic acid binding/elution conditions.
• Evaluation of reducing agents, chaotropic salts, heat, proteinase K, and elution buffers.
• Assessment of downstream application compatibility using loop-mediated isothermal amplification (LAMP).

Main Results:

• The POC-Pure method efficiently purifies and concentrates both DNA and RNA from saliva.
• Achieved a low limit of detection: under 0.25 copies/μL for DNA and 0.5 copies/μL for RNA with 200 μL input.
• Demonstrated successful on-chip nucleic acid extraction and purification.
• Validated downstream application compatibility with POC-suitable LAMP.

Conclusions:

• Developed and demonstrated a simplified on-chip nucleic acid extraction method from saliva.
• POC-Pure offers a cost-effective and efficient solution for point-of-care molecular diagnostics.
• The method addresses key challenges in saliva-based nucleic acid purification for POC applications.