RHEONIX ADVANTAGES

The Rheonix CARD™ is extremely versatile and outperforms its labor-intensive "bench-top" counterparts.

CARD™ advantages:

  • Virtually no hands-on effort results in significantly reduced labor costs.
  • Reduced sample volumes and reagent volumes save time and money.
  • Sophisticated assays can be performed by almost anyone since all steps are automatic.
  • Portability allows accomplishing assays in "point-of-care" or "point-of-use" settings.

Performance Test 1 — Detection of Specific Gene Sequences:
A raw clinical whole blood specimen was introduced onto the CARD™, which had been programmed to perform all steps required by an independent testing laboratory to evaluate for the presence of β - thalassemia gene markers. The only hands-on effort required was the initial introduction of the whole blood sample while all other steps were performed automatically. Manual performance of the assay on the bench top required the lysing of the cells, extraction and purification of DNA, introduction of PCR primer pairs and reaction mixture to the samples, and, then, placement within a bench-top thermocycler. The comparison of two separate clinical specimens revealed the expected 660 bp amplicons in all samples (Figure 1). Although the bench-top signals appear stronger, the volume of raw blood analyzed was 150 µL while the volume of sample applied to the CARD™ was only 25 µL. Time-lapse photography of the CARD™ shows the change in color as the whole blood is lysed (A and B) and then moves through the DNA isolation components of the CARD™ (C). Side view of CARD™ mounted in controller (photo left).

Figure 1
Whole Blood PCR Analysis

β - Thalassemia Results:
Lane 1 (molecular weight); Lanes 2 and 3 were entirely performed on a Rheonix CARD™ while Lanes 4 and 5 were entirely manually performed on the bench top. Lanes 2 and 4 were obtained from the same individual, and Lanes 3 and 5 were obtained from a second individual. The starting volume of the bench top PCR analysis was 200 µL while that used in the Rheonix CARD™ was only 50 µL.

Performance Test 2 — HPV Detection & Reverse Dot Blot Analysis:
The Rheonix CARD™ was designed and programmed to perform all steps required to analyze vaginal swabs for the presence of the various subtypes of Human Papilloma Virus (HPV). Once the swabs were obtained, they were placed into a tube of PBS and mixed to release any virions. The only other manual effort required was the initial introduction of the samples to the CARD™. All of the following subsequent steps were performed automatically with no intervention:

  • Lysis of virions
  • Extraction/Purification of DNA
  • PCR amplification, using 5'-biotinylated primer pairs
  • Denaturation of double-stranded biotinylated PCR amplicons
  • Introduction of biotinylated, single-stranded PCR amplicons to an array of capture probes specific for the various subtypes of HPV
  • Washing steps
  • Introduction of streptavidinylated horseradish peroxidase and subsequent washing steps
  • Introduction of TMB substrate

The individual components of the Rheonix CARD™ were designed to seamlessly perform the required steps (Figure 2.) Unequivocal results were obtained when two separate clinical specimens were analyzed for the presence and identity of HPV.

Figure 2
Fully-Integrated Detection of Human Papilloma Virus

Results: Human vaginal swabs were analyzed in a fully-integrated manner. All sample preparation, analysis, and readout functions, using a reverse dot blot array, were performed automatically on the CARD™ for two separate clinical specimens.