An inkjet-printed polysaccharide matrix for on-chip sample preparation in point-of-care cell counting chambers

Abstract

On-chip sample preparation in self-contained microfluidic devices is a key element to realize simple, low-cost, yet reliable in vitro diagnostics that can be carried out at the point-of-care (POC) with minimal training requirements by unskilled users. To address this largely unmet POC medical need, we have developed an optimized polysaccharide matrix containing the reagents which substantially improves our fully printed POC CD4 counting chambers for the monitoring of HIV patients. The simply designed counting chambers allow for capillary-driven filling with unprocessed whole blood. We carefully tailored a gellan/trehalose matrix for deposition by inkjet printing, which preserves the viability of immunostains during a shelf life of at least 3 months and enables controlled antibody release for intense and homogeneous immunofluorescent cell staining throughout the complete 60 mm² image area within 30 min. Excellent agreement between CD4 counts obtained from our fully printed CD4 counting chambers and the gold standard, flow cytometry, is demonstrated using samples both from healthy donors and HIV-infected patients.

Fig. 1. Illustration of the on-chip sample preparation approach. (a) Perspective view of a simple glass chip with two chambers. Each chamber (some tens of μm high) has two openings to enable capillary flow. Purple shading indicates reagent concentration. (Left) In a (hypothetical) chamber with reagents deposited without matrix, reagents would be washed off toward the end of the chamber during inflow. (Right) In a chamber with reagents embedded in a matrix, reagents are homogeneously distributed, as their release is delayed during inflow. (b) (Side view) (Left) The chamber without matrix shows strong reagent wash-off. (Right) The chamber with matrix shows uniform reagent distribution. Purple shading indicates reagent concentration.

Fig. 2. (a) Correlation of CV of roughness and CV of fluorescence intensity (APC-αCD3 readout) in polysaccharide/Ab layers. (b) Normalized and background corrected intensity ratios, along inflow direction, indicating the distribution of APC-αCD3 in the counting chambers with different cast layers after/before sample inflow. (c) Representative scatter plots and histograms obtained using our image cytometry method. Plotted are the fluorescence intensity (I) of APC-αCD3 (red excitation) and PerCP-αCD4 (blue excitation) stained cells in counting chambers with: prestained cells (left), cells stained in chambers with cast gell/Ab layers (center) and cells stained in chambers with cast chi/Ab layers (right), all after 30 min incubation. Purple dots are cells identified as the double positive cell population.

Fig. 3. (a) Fluorescence intensities (I) of dry printed layers with varying fractions of trehalose but equal amounts of gell and fluorophore labeled Ab. (b) The intensity and thickness (d) ratios of the layer after and before PBS immersion. Data points represent mean ± standard deviation (n = 4). (c) Average intensities (PerCP vs. APC readout) obtained using our image cytometry method of CD4+ T-cells stained in counting chambers containing printed gell/Ab layers with varying trehalose contents. Data points represent mean ± standard deviation (n = 3). (d) Summary of average intensities (PerCP vs. APC readout) obtained using our image cytometry method of CD4+ T-cells at “filling” vs. “venting regions” stained in counting chambers containing printed gell/Ab layers with varying trehalose contents. Data points represent mean ± standard deviation (n = 4). The dashed diagonal line represents the ideal case, where the average intensities of stained cells located at the “filling region” and the “venting region” are identical, which implies the absence of Ab wash-off. (e–g) Scatter plots and histograms of fluorescence intensities obtained using our image cytometry method of APC-αCD3 (red excitation) and PerCP-αCD4 (blue excitation) stained cells in counting chambers containing: (e) pure gell (no trehalose), (f) gell/trehalose (17%) and (g) gell/trehalose (33%) matrices. Black solid circles represent all double (CD3 and CD4) positive cells (i.e. CD4+ T-cells) found in the entire image area.

Fig. 4. (a) The ratio of fluorescence intensity (I) in both APC (red squares) and PerCP (blue crosses) readouts of dry printed layers after storage over silica gel at 4 °C (4sg) and the intensity of comparable fresh layers. Data points represent mean ± standard deviation (n = 4). (b) Comparison between CD4 counts (#cells per μL) of whole blood from healthy donors, measured using our image cytometry approach and standard flow cytometry. For each sample with a given leukocyte concentration, 4 separate counting chambers were used and 3 separate flow cytometry analyses were performed. The averages of the flow cytometry results were plotted against the individual counts using our image cytometry approach.

Fig. 5. (a) Comparison between CD4 counts (number of CD4⁺ T-cells per μL) of HIV-infected patients obtained with standard flow cytometry and with image cytometry using fully printed CD4 counting chambers, with the linear regression demonstrating excellent agreement. (b) Bland–Altman plot of the same data.