A simple and sensitive transient ITP method for on-chip analysis of PCR samples

Area of Science:

• Analytical Chemistry
• Microfluidics
• Biotechnology

Background:

• Polymerase chain reaction (PCR) generates complex, highly saline samples.
• Direct analysis of PCR samples is challenging due to matrix effects and low analyte concentrations.
• Existing preconcentration and separation methods can be time-consuming and complex.

Purpose of the Study:

• To develop a sensitive, simple, and robust on-chip method for analyzing PCR samples.
• To improve detection sensitivity and reduce analysis time for PCR products.
• To enable direct analysis of complex PCR samples without extensive sample preparation.

Main Methods:

• On-chip transient isotachophoresis (TITP) on a poly(methyl methacrylate) microchip.
• Sample injection into a free-solution buffer channel for increased loading and reduced bias.
• Stacking of sample ions using chloride as leading ions and HEPES as terminating ions.
• Separation of stacked plug by zone electrophoresis (ZE) in hydroxypropylmethyl cellulose.
• Continuous operation with sequential voltage switching in a single background electrolyte.

Main Results:

• Achieved a 20-fold signal enhancement compared to conventional methods.
• Obtained an average limit of detection of 0.24 pg/µL (S/N = 3).
• Demonstrated improved detection sensitivity without loss of resolution.
• Successfully analyzed highly saline PCR samples directly.

Conclusions:

• The developed TITP method is sensitive, simple, and robust for on-chip PCR sample analysis.
• This method offers significant advantages for direct analysis of complex biological samples.
• The TITP technique is well-suited for high-throughput and point-of-care applications.