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. 2012 Mar 1;11(1):42-51.
doi: 10.1097/POC.0b013e318222e184.

Rheonix CARD(®) Technology: An Innovative and Fully Automated Molecular Diagnostic Device

Gwendolyn Spizz  1 , Lincoln Young, Rubina Yasmin, Zongyuan Chen, Travis Lee, Deborah Mahoney, Xun Zhang, Greg Mouchka, Benjamin Thomas, Whitney Honey, Todd Roswech, Cristina McGuire, Richard Montagna, Peng Zhou
Affiliations

Rheonix CARD(®) Technology: An Innovative and Fully Automated Molecular Diagnostic Device

Gwendolyn Spizz et al. Point Care. .

Abstract

A versatile microfluidic platform for the evolving molecular diagnostics industry is described. It incorporates low cost Rheonix CARD(®) (Chemistry and Reagent Device) technology to analyze a variety of clinical specimens. A patented lamination process incorporates all pumps, valves, microchannels and reaction compartments into an inexpensive disposable plastic device. Once an untreated clinical specimen is introduced, all assay steps, including cell lysis, nucleic acid purification, multiplex PCR, and end-point analysis, are automatically performed. Three distinct CARD assays are described which utilize either a low density microarray for multiplex detection of amplicons or an integrated primer extension assay to detect single nucleotide polymorphisms of interest. The STI (Sexually Transmitted Infections) CARD(®) is able to simultaneously detect four sexually transmitted infectious agents (N. gonorrhoeae, C.trachomatis, T. pallidum and T. vaginalis). Human C33A cervical epithelial cells were spiked with different levels of genomic DNA from the four species of interest, singly or in combination, and applied to the CARD device. Using multiplex PCR amplification of the targets followed by microarray detection, the CARD device was able to correctly detect a minimum of 10 copies of each of the four pathogens. The HPV (Human Papillomavirus) CARD(®) was able to detect and distinguish 20 different clinically relevant HPV types using cloned HPV DNA. In addition, the HPV CARD could identify HPV types in vaginal specimens previously demonstrated to contain high or low risk HPV using a currently commercially available testing method. Finally, the detection of specific single nucleotide polymorphisms (SNP) associated with warfarin dosing sensitivity was achieved on the Warfarin Genotyping CARD(®) by analyzing human buccal swabs. Once multiplex PCR was completed, the SNPs were detected using a primer extension assay.

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Figures

Figure 1
Figure 1. Pump/Valve Assembly on Rheonix CARD® device
The polystyrene CARD® consists of a 1 mm upper layer and a 1 mm lower layer that are laminated together, with a 38 μm polystyrene layer between. Positive pressure through the aperture in the diaphragm pocket of the lower layer causes the deformable membrane to be driven upward, thereby preventing flow of fluids from one discontinuous channel to the next. Conversely, negative pressure causes the deformable membrane to be drawn down into the diaphragm pocket, thus allowing fluid to flow from one discontinuous channel to the next. In addition to serving as either open or closed valves as depicted, the same diaphragm pockets can serve as pumps by sequential application of positive and negative pressure.
Figure 2
Figure 2. Rheonix Quad CARD® Configuration
A Rheonix Quad CARD® is shown that is capable of performing up to four simultaneous molecular assays. Once the biological sample is applied where noted, the cells are lysed and DNA purified through an onboard silica membrane. The isolated DNA obtained from each individual sample can then be delivered to two separate PCR thermocycling chambers to overcome potential inhibition from multiple primer pairs required to PCR amplify multiple targets. Once PCR has been completed, the amplicons generated in each of the two PCR thermocycling chambers can be combined and delivered to the terminal portion of the CARD where the reverse dot blot or primer extension assays are performed.
Figure 3
Figure 3. Reproducibility of Rheonix Quad CARD®
As described in the text, 30 μl aliquots of a cell suspension were added to each of the four sample reservoirs of a Quad CARD® and DNA automatically isolated. (A) The spectrophotometric profile of each of the four isolated DNA samples was analyzed. An aliquot of the isolated DNA from one channel was used to PCR amplify the target sequence in a conventional bench top thermocycler while DNA from each of the other three quadrants was automatically PCR amplified on the CARD®. (B) Agarose gel electrophoresis of the amplicons generated using a bench top theromocycler (BT) and the amplicons generated automatically in each of the two thermocycling chambers of the three remaining quadrants on the CARD® (CARD®)
Figure 4
Figure 4. Rheonix STI CARD® Assay
Five million C33A cells/ml were spiked with 10,000 copies/ml each of genomic DNA from N. gonorrhoeae, C. trachomatis, T. denticola, and T. vaginalis, and subjected to Rheonix STI CARD® assay as described. Left panel: Image shows the colored precipitate formed from HRP activity following the capture of biotinylated amplicons incubated with strepdavidinylated HRP and substrate. Right panel: Filter key; SC: Spotting Control- biotin labeled poly-A; CT: C. trachomatis capture probe; TP: T. pallidum capture probe; TV: T. vaginalis capture probe; and NG: N. gonorrhoeae capture probe. Note that T. denticola is recognized by the T. pallidum probe in this assay. (B) Five million C33A cells/ml were spiked with (1) no STI DNA, or 40,000 copies/ml of genomic DNA from (2) T. denticola, (3) N. gonorrhoeae, C. trachomatis, and T. vaginalis, or (4) all 4 microbial DNAs and subjected to Rheonix STI CARD® assay as described. Filter key is the same as shown in Figure 4A
Figure 5
Figure 5. HPV CARD® Analysis of cloned plasmid DNA
Reverse dot blot results obtained from processing C33A human cells spiked with various HPV plasmids as described in text. (B) Filter key for probe spots. SP refers to a spotting control consisting of biotinylated poly A to demonstrate effective spotting of probes and performance of the HRP color generating reaction. Gb corresponds to human beta-globin capture probe. All numbers pertain to the HPV type of the individual probes. All probes except Gb, HPV 16, and 18 were spotted at 50 μM. Gb, 16, and 18 were spotted at 2 (L), 20 (M), and 50 (H) μM. SC spotted at 2.5 μM. Note, the presence of HPV 56 detected on HPV 66 hybridized membrane is due to mixed HPV 56/66 clone, and not cross-hybridization.
Figure 6
Figure 6. HPV CARD® Results of authentic clinical specimens
Reverse dot blot results obtained from evaluating 15 clinical specimens previously shown to be positive using the HC2 Hybrid Capture assay. (B) Reverse dot blot results obtained from evaluating 6 clinical specimens previously shown to be negative using the HC2 Hybrid Capture assay. Both sets of experiments were performed as described in the text. C) Membrane key for 9015, 8992, 9016, 8987, 9003, 8988, and 9025 (italics). D) Membrane key for all others. Membrane keys. SP: Spotting Control; biotin labeled poly-A. All probes spotted at 20 μM; Globin (Gb), 16 and 18: spotted at 0.2 (L), 2 (M), and 20 (H) μM.
Figure 7
Figure 7. Representative image derived from the primer extension component of the Warfarin Genotyping CARD®
Warfarin Genotyping was performed as described in the text. (A) The key indicates the allele targeted by the capture probe (NC = noncoding strand, C = coding strand, na = not applicable). (B) Results of seven individual genotypes obtained using the Rheonix Warfarin Genotyping CARD®. Filter results from buccal swabs of seven individuals are shown in the lower portion of the (B). The upper portion of (B) indicates the genotypes read from these filters. In addition, all genotypes were verified via bi-directional sequencing. Note these genotypes were represented within the 20 individuals tested. The more rare combinations of genotypes were not expected in this small sample set. Current experiments are underway to validate the assay with the more rare genotypes.
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