An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids

Abstract

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids.

Fig. 1

A schematic of the polycarbonate fluidic chip consisting of reagents storage pouches (P1-P6), on-chip diaphragm valves (V1-V4), a mixing chamber, a nucleic acid isolation chamber housing a silica membrane, a (RT-)PCR chamber preloaded with dried reagents, an amplicon dilution trap, a waste chamber and liquid trap, and a lateral flow strip. (a) Top view; (b) cross-sections along the length of the cassette (indicated with the arrows B-B in A) showing the cassette in storage state (B1) and operating (after activation) state (B2). Note that heaters H1 and H2 are components of the analyzer but not of the cassette. ‘TE’ denotes the thermoelectric modules

Fig. 2

A photograph of the assembled, nucleic acid cassette in its storage state. For better visibility, the various storage pouches were filled with dyes. Pouch P1 (100 μl) contains the binding/lysis buffer; pouch P2 (60 μl) contains the inhibitor removal buffer; pouch P3 (100 μl) stores the wash buffer; pouch P4 (40 μl) contains the elution buffer; pouch P5 (60 μl) contains the lateral flow buffer; and pouch P6 (60 μl) contains a suspension of UCP reporter particles

Fig. 3

A schematic depiction of the analyzer comprising linear actuators, a pair of thermoelectric (TE) units and a heat sink, a K-type thermocouple, a micro-vacuum pump, a liquid trap, and a microcontroller. All the components are packaged in a portable box. The external computer (PC) provides user interface and data analysis software

Fig. 4

Schematic of the on-chip pumping and valving. (a) Fluid stored in the pouch is “excreted” out through the pouch's exit port when the plunger deforms the pouch membrane. The pouch's volume determines the discharged fluid's volume, and the actuation speed determines the dispensing rate. (b) Actuation of the on-chip, normally open diaphragm valve. The vertical movement (in Z direction) of the piston is generated with a linear actuator

Fig. 5

(a) A schematic of the on-chip mixing chamber. The mixing process is enhanced with a low-power, high frequency, miniature, vibrating disk motor (VDM) placed next to the bottom surface of the mixing chamber (purple). The VDM is housed in the analyzer. (b) Image b1 features two distinguishable fluids (red and green) at the time of their introduction into the mixing chamber; b2 shows the two fluids after 15 s of diffusion; b3 shows the mixture of the two fluids after 15-seconds of stirring with the VDM. (The dashed line in Figs. b1 and b2 delineates the interface between the two dyes for the benefit of readers who do not have access to color images)

Fig. 6

The mass loss (%) of pouch-stored liquids water (upright triangles) and ethanol (squares), as functions of time under room conditions

Fig. 7

(a) The PCR chamber's temperature as a function of time. (b) An enlarged segment of (a) spanning the time interval from 80 s to 200 s (b) and depicting, respectively, the temperatures at the outer bottom surface in contact with the TE unit (dotted line) and the top outer surface in contact with the TE unit (dashed-dot line), inside the reactor (solid red line), and the set temperature (solid line) as functions of time

Fig. 8

(a) A photograph of the modular PCR chamber with the paraffin-encapsulated, pre-loaded, dry reagents. (b) Cross-section of a reactor with the paraffin-encapsulated, pre-loaded, dry reagents. (c) Agarose gel (1.5%) electropherograms of PCR products (305 bp) amplified from ∼1.5 ng B.Cereus DNA (30 cycles). Lanes 1 and 2 correspond, respectively, to the products of the PCR reactor with liquid reagents and the PCR reactor with wax-encapsulated dry reagents. (d) Agarose gel (2%) electropherograms of RT-PCR products (155 bp) amplified from armored RNA isolated from a 100-μl sample of 1×10⁵ virions/ml (35 cycles). Lanes 1, 2 and 3 correspond, respectively, to the products of the RT-PCR reactor with liquid reagents, the reactor with dry reagents, and the negative control (no target) with dry reagents

Fig. 9

(a) Schematic depiction of the silica membrane isolation chamber. (b) The DNA concentration C(n) of consecutive eluted aliquots (n) from the on-chip silica membrane; the error bar represents the scatter of data from three independent measurements. (c) Real-time PCR of samples taken from the consecutive aliquots: the fluorescent intensity (arbitrary units) is depicted as a function of time

Fig. 10

(a) Scan of processed saliva-spiked with B. Cereus (1.4×10⁴ cells/mL) sample. The emission intensity (in relative fluorescent units) is depicted as a function of position along the strip. (b) The ratio of the test and the control signals (T/C) of saliva samples spiked with B. Cereus as a function of cell concentration (cells/mL)

Fig. 11

The ratio of test and control peaks as a function of armored RNA virion concentration

Fig. 12

The ratio of test and control peaks as a function of HIV concentration