On-chip microtubule gliding assay for parallel measurement of tau protein species

Abstract

Tau protein is a well-established biomarker for a group of neurodegenerative diseases collectively called tauopathies. So far, clinically relevant detection of tau species in cerebrospinal fluid (CSF) cannot be achieved without immunological methods. Recently, it was shown that different tau isoforms including the ones carrying various types of mutations affect microtubule (MT)-kinesin binding and velocity in an isoform specific manner. Here, based on these observations, we developed a microfluidic device to analyze tau mutations, isoforms and their ratios. The assay device consists of three regions: a MT reservoir which captures MTs from a solution to a kinesin-coated surface, a microchannel which guides gliding MTs, and an arrowhead-shaped collector which concentrates MTs. Tau-bound fluorescently labeled MTs (tau-MTs) were assayed, and the increase in fluorescence intensity (FI) corresponding to the total number of MTs accumulated was measured at the collector. We show that our device is capable of differentiating 3R and 4R tau isoform ratios and effects of point mutations within 5 minutes. Furthermore, radially oriented collector regions enable simultaneous FI measurements for six independent assays. Performing parallel assays in the proposed device with minimal image processing provides a cost-efficient, easy-to-use and fast tau detection platform.

Fig. 1

Schematic representation of a) Microfludic device comprising of MT reservoir, microchannel and MT collector. Angles of the MT collector, a = 20° and b = 55°, are defined as shown in the enlarged schematic. b) Preparation of tau-MT. c) Overview of the six assay units radially patterned for simultaneous measurement of MT accumulation at all the six collectors.

Fig. 2

Microfluidic device fabrication and Pluronic surfactant treatment.

Fig. 3

MT motility and accumulation in the collector region in a microfluidic device. a) Sequential images of a MT gliding in a channel towards collector. b) MTs recirculated within collectors. c) MTs concentrated in collectors at 15, 45 and 90 min. Scale bar, 20 µm. d) FI of 2N4R-MT and no tau-MT. 2N4R-MT collector showed a significantly lower FI (t-test: p < 0.001 mean ± SEM), and difference was significant 5 min after introducing MTs. Inset represents the FI profile of no tau-MT at the collector for 90 min.

Fig. 4

Differentiation of different 2N3R:2N4R isoforms. FIs decreased with the increase of 2N4R tau. Mean ± SEM; ***: p < 0.001; *: p < 0.05; n.s.: p > 0.05 (ANOVA); n ≥ 12.

Fig. 5

Various 2N4R tau mutations demonstrate distinct effects on FIs. MTs incubated with wild 2N4R showed consistently lower FI than any of the five mutants analyzed. FI for R406W was significantly lower than those for V248L, G272V, V337M and P301L (p < 0.05). FI for G272V was significantly lower than those for V337M and P301L (p < 0.05) and higher than those for V248L and R406W (p < 0.05), respectively. Mean ± SEM; *: p < 0.05; n.s.: p > 0.05 (ANOVA); n ≥ 16.

Fig. 6

Detection limit of the device. Significantly lower MT accumulation became apparent above 100 nM 2N4R. No significant difference with a control (no tau-MT) was noticed for 10 nM 2N4R. Mean ± SEM; *: p < 0.05; n.s.: p > 0.05 (ANOVA); n ≥ 16.