Eliminating viscosity bias in lateral flow tests

Abstract

Despite the widespread application of point-of-care lateral flow tests, the viscosity dependence of these assay results remains a significant challenge. Here, we employ centrifugal microfluidic flow control through the nitrocellulose membrane of the strip to eliminate the viscosity bias. The key feature is the balancing of the sample flow into the cassette of the lateral flow test with the air flow out of the cassette. A viscosity-independent flow rate of 3.01 ± 0.18 µl/min (±6%) is demonstrated for samples with viscosities ranging from 1.1 mPas to 24 mPas, a factor greater than 20. In a model human IgG lateral flow assay, signal-intensity shifts caused by varying the sample viscosity from 1.1 mPas to 2.3 mPas could be reduced by more than 84%.

Keywords: Engineering; Physics.

Fig. 1. Schematic illustration of the working principle of the centrifugal cassette from no flow to a constant flow rate Q_s.

a Schematic illustration of the microfluidic design inside the cassette. b The rotational frequency is accelerated to 15 Hz. The sample starts flowing into the pneumatic chamber with the membrane inside. This sample inflow causes an overpressure that acts against the sample inflow. With increasing overpressure in the chamber, air starts flowing out of the pneumatic chamber through the venting resistance channel. c At equilibrium, the flow rates of both the sample inflow and the air outflow through the resistance channel are equal and the overpressure in the chamber remains unchanged. This is the actual operating state of the structure

Fig. 2. Overview of the centrifugal cassette with pneumatic flow control.

a Schematic illustration of the structure with all relevant parameters. b Left: Structure overview; the optional vents were not used for the experiments in this study. Right: Stroboscopic picture of the thermoformed cassette during the experiment at 15 Hz

Fig. 3. Measured flow rates of samples with viscosities of 1.1 mPas, 2.3 mPas, and 24 mPas at 15 Hz.

The mean value is indicated with a line, and the density-dependent expected flow rate for each sample is shown with a dashed line. Each gray circle represents a measured value

Fig. 4. Schematic concept of the model human IgG lateral flow assay.

a Without analyte (human IgG) in the sample, the conjugated AuNPs only bind to the human IgG on the test line. b With the analyte in the sample, the conjugated AuNPs also binds to human IgG in solution, and less conjugated AuNPs binds to the test line. Hence, the test line shows a weaker signal

Fig. 5. Scanned test lines of lateral flow strip assays that were performed as dipstick assays (a) and in the centrifugal cassette (b).

The quantitative analyses of all test-line signal intensities are shown below. For better comparison, the signal intensity is defined as the relative intensity compared to the signal intensity of the negative control with a sample viscosity of 1.1 mPas. a The increase in the viscosity of the sample resulted in a mean signal increase of 38 percentage points. This difference can be seen with the naked eye, especially at high and low concentrations. b Using the centrifugal cassette, the mean signal increase was reduced to only six percentage points