A portable, integrated analyzer for microfluidic - based molecular analysis

Abstract

A portable, fully automated analyzer that provides actuation and flow control to a disposable, self-contained, microfluidic cassette ("chip") for point-of-care, molecular testing is described. The analyzer provides mechanical actuation to compress pouches that pump liquids in the cassette, to open and close diaphragm valves for flow control, and to induce vibrations that enhance stirring. The analyzer also provides thermal actuation for the temperature cycling needed for polymerase chain reaction (PCR) amplification of nucleic acids and for various drying processes. To improve the temperature uniformity of the PCR chamber, the system utilizes a double-sided heating/cooling scheme with a custom feedforward, variable, structural proportional-integral-derivative (FVSPID) controller. The analyzer includes a programmable central processing unit that directs the sequence and timing of the various operations and that is interfaced with a computer. The disposable cassette receives a sample, and it carries out cell lysis, nucleic acid isolation, concentration, and purification, thermal cycling, and either real time or lateral flow (LF) based detection. The system's operation was demonstrated by processing saliva samples spiked with B. cereus cells. The amplicons were detected with a lateral flow assay using upconverting phosphor reporter particles. This system is particularly suited for use in regions lacking centralized laboratory facilities and skilled personnel.

Fig. 1:

Microfluidic cassette for nucleic acid processing. Left: A photograph of the cassette. Right, top: A schematic depiction of the pouch actuation. Right, bottom: A schematic depiction of the valve actuation.

Fig. 2:

The portable nucleic acid diagnostics system with a pulled out drawer

Fig. 3:

The portable nucleic acid diagnostic system – folded model.

Fig. 4:

Active, double-sided heating system

Fig. 5:

A block diagram of communications and control

Fig. 6:

The set (desired) PCR temperature (thin solid line), the temperature of the bottom heated surface (sparse dotted line), the temperature of the top heated surface (densely dotted line), and the temperature inside the cassette (thick solid line) as functions of time during one heating cycle

Fig. 7:

Electropherograms of amplicons of B. cereus bacterium DNA. Lane 1 is a marker VIII ladder. Lanes 2 and 3 correspond, respectively, to the bench-top and cassette’s PCR products.

Fig. 8:

(a) Lateral flow strip. (b) The emission intensity of the UCP reporter particles as a function of position along the lateral flow strip. (Top) Positive sample of saliva spiked with 1.4 × 10⁴ B. Cereus cells. (Bottom) Negative control (no cells). (c) The ratio of the areas under the test and the control peaks (T/C) of saliva samples spiked with B. Cereus as a function of cell concentration (cells/ml).