May 1, 2015 (Vol. 35, No. 9)

Varoujan D. Amirkhanian Director BiOptic
Shou-Kuan (Eric) Tsai President BiOptic
Zsolt Ronai M.D., Ph.D. Professor and Research Scientist Semmelweis University
Gene W. Stewart President BiopHoretics

A Novel Single-Channel Microfluidic Device for Nucleic Acid Fragment Analysis

Nucleic acid analysis, genotyping, PCR, and RFLP methodologies are time-consuming techniques using agarose or acrylamide gel electrophoresis—image capture plus gel image interpretations. Capillary gel electrophoresis (CGE) automates these, providing fast separations with high sensitivity, excellent resolution, and ease of operation. However, despite its effectiveness current CGE  has a high cost of instrumentation and cost per sample. Much of this cost is due to traditional designs.

BiOptics’ Qsep100™ CGE system solves this problem by integrating microcapillary electrophoretic technology into a plug-and-play disposable pen-shaped capillary gel cartridge with automated liquid handling, real time fluorescent detection, and analysis. Optimized for high resolution and high throughput at ambient temperatures, the need for expensive, extreme voltage power supplies and external cooling is eliminated, thus reducing instrument costs and increasing productivity.

Genotyping and other relevant applications with sample throughputs to 96 can be automatically analyzed within 5–6 hours. By integrating and automating all analytical functions required for the various types of nucleic acid analysis, Qsep100 provides improved peak resolution, good linear dynamic range, and reproducible migration times with a lowered cost. 


System Design Overview

Qsep100 was designed to integrate, automate, and miniaturize the laborious techniques of traditional DNA electrophoresis, and reduce the costs associated with traditional CGE.

The Qsep100 CGE system (Figure 1) is composed of the core instrument (38 x 39 x 40 cm), an automated liquid/sample handling (X-Y-Z) platform, real-time fluorescence detector, and multi-use disposable pen-shaped capillary gel-cartridges; all are driven by an intuitive GUI with analytics. The platform contains buffer(s), a sample waste reservoir, DNA ladder(s), and samples arrayed in a 96-well format. This design provides flexibility in single-channel CGE instrument applications at low cost-per-sample run analysis of DNA fragments.

The unique pen-shaped capillary gel-cartridge (CGC) permits plug-and-play use in a robust, injection-molded body with an integrated gel-ethidium bromide reservoir and a short length single microfluidic glass capillary column (75 µm ID, 15 cm total length, 11 cm separation length).

The shortened capillary length allows for reduced operating voltages (1–15 KV), shorter run times, and the elimination of expensive cooling systems such as Peltier or recirculating chillers. To mitigate electroosmotic flow (EO) and prevent analyte adhesion to the inside walls of the capillary column, they are dynamically coated with the separation matrix.


Figure 1. (A) BiOptic’s Qsep100. (B) The Q-Analyzer software with easy-to-use GUI for easy operation and rapid data analysis. (C) Multi-use disposable pen-shaped capillary gel cartridge.

This design also includes top and bottom electrodes (anode and cathode), a detection window, imbedded radio-frequency identification (RFID) chip, and a label to identify the gel-cartridge and monitor its number of electrophoretic runs.

Each cartridge contains a proprietary gel/ethidium bromide mixture and is capable of analyzing 200 samples in 2 minutes per sample analysis, consuming as little as 1 pl from sample volumes between 1 µl to 20 µl. 

Real-time sample detection is achieved by an innovative optical fiber system (Figure 2) with micro-ball ended fibers for emission and excitation. These provide for higher detection sensitivities (0.1 ng/µl) and significantly reduced background noise (for improved signal to noise ratio). This system uses a low cost, powerful light-emitting diode (LED) induced fluorescent detection system to excite at 520 nm and detects 590–650 nm emission wavelengths.


Figure 2. (A) Photograph of the ball-end dual fiber optic system. (B) Schematic of the LED-induced fluorescence detection system with pneumatically operated fork assembly, which aligns two optical fibers precisely to the detection zone of the glass capillary by clamping to the two pre-installed plastic ferrules (excitation @ 530 nm; emission detection @ 590–620 nm).

Simple System Operation

Buffer(s), markers, and samples are placed in the buffer/sample tray within the instrument and the capillary gel cartridge is inserted (Figure 3). Within the software, the user selects their preprogrammed method or creates a new program. The user enters run parameters and sample locations within the 96-well sample tray and begins the analysis.  The Q-Analyzer software automatically calculates the size of detected fragments (bp) using a reference DNA Ladder.


Figure 3. (A) Installation of the capillary gel-cartridge. (B) Buffer solution and ladder tray with 96-well sample tray.

Proofs

Qsep100 was evaluated for use in genotyping fragment analysis and for its limit of detection and linear performance and resolution (Figure 4).

Figure 4A shows the limit of detection (LOD) using a 576 bp PCR DNA fragment diluted in water vs. a sizing ladder. Sample concentrations range from 0.002 ng/µL to 0.01 ng/µL.

Figure 4B shows linearity of detection with the same 576 bp PCR fragment serially diluted in a sample buffer. Separation conditions for Figures 4A and 4B are an injection voltage of 4 kV/10 s, a separation voltage 6 kV at ambient temperature.

Figure 4C demonstrates the capability of the PCR and RFLP methodologies in determining co-infections of different human papilloma virus (HPV) types. The sample was determined to have a high risk genotype HPV (type 66) and a low risk genotype HPV (type 6). The conditions are an injection at 4 KV/4sec and a separation at 8 KV/220 seconds at ambient temperature.

Figure 4D shows the resolution power of the system.


Figure 4. (A) Limit of detection. (B) Linearity of performance. (C) Genotyping results showing the capability of the PCR/RFLP methodology in determining co-infections of different HPV types. (D) Resolution.

Summary

The Qsep100 CGE system is a robust, easy-to-use, cost-effective, and efficient alternative to traditional CGE and slab gel electrophoresis for DNA fragment analysis.  Its unique multi-use, pen-shaped, capillary gel cartridge coupled with real-time LED-induced fluorescence detection eliminates the need for expensive cooling systems, maintains  high separation performance, and reduces instrument and sample analysis costs.

PCR and RFLP genotyping can be performed in an automated fashion at higher speeds (90% time saving) and lower costs ($0.11 to $0.20 per sample).

With demonstrated resolution as low as 2–4 base pairs, a limit of detection as low as 0.1 ng/µl, and a linear performance of R2= 0.9997, it has been shown that Qsep100 is a powerful and cost-effective tool for genotyping applications including restriction digests, PCR product analysis, and total RNA and plasmid purification for both low- and high-throughput DNA and RNA research facilities.


























Varoujan D. Amirkhanian (varoujamirkhanian@bioptic.com.tw) is director and Shou-Kuan (Eric) Tsai (eric.tsai@bioptic.com.tw) is president at BiOptic, Zsolt Ronai M.D., Ph.D., is professor and research scientist at Semmelweis University, and Gene W. Stewart (gstewart@biophoretics.com) is president at BiopHoretics. Note: Qsep100 DNA Fragment Analyzer is distributed by BiopHoretics.

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