The primary barriers to multiplexed point of care immunoassays are: (1) cost; (2) response time; and (3) sample handling. Described here is a self-contained, multiplexed immunoassay platform for point of care detection that leverages a number of enabling technologies to address these barriers. This platform is referred to as the ;;D4;; assay, as it is composed of the following four sequential, concerted events (Figure 1): (1) Dispense (droplet of blood); (2) Dissolve (printed reagents on chip); (3) Diffuse across surface; (4) Detect binding event.
The D4 assay process begins when a finger-stick is administered and the resulting droplet of blood is applied to the surface of a detector chip. Hydrophobic ink printed onto the surface of the chip confines the blood droplet to a non-fouling region containing soluble, labile spots of detection antibodies and insoluble, non-labile spots of capture antibodies. As the soluble detection antibodies are dissolved from their printed spots by the droplet of blood, three serial events occur to generate signal (Figure 2): (1) the first half of the detection complex is formed by the binding of analytes present in blood to the stable capture agent spots; (2) diffusion of the blood laterally through the polymer brush, resulting in the dissolution and diffusion of soluble detection antibody spots; (3) solubilized detection antibodies bind to their respective analyte-capture agent spots, completing the detection complex and resulting in signal generation at the position of the non-labile capture antibody spots.
This assay relies upon the ability of labeled detection antibodies, printed into a nonfouling brush as ;;labile spots;;, to be carried by blood flow to adjacent rows of stably immobilized capture antibodies by diffusion of the analyte solution (Figure 2). Generation of signal at a given capture spot location provides identification of individual analytes (positives). Quantification of the concentration of the different analytes is carried out identically to a conventional fluorescence immunoassay by pre-calibration of the system using a dilution series of the analyte spiked into whole blood.
The D4 assay addresses several critical needs in point of care testing as follows: First, the cost of testing is reduced through miniaturization, multiplexing and one-step, on-site processing of undiluted whole blood obtained from a finger stick. Second, in order to simplify the immunoassay process, the D4 relies on diffusion to bring spatially localized reagents together to create a functional assay and thereby eliminate the need for liquid transfer steps, microfluidic manipulation of sample or reagents, and wash steps. Third, this multiplexed platform is capable of screening for a panel of markers in a single drop of blood with no sample preprocessing. Fourth, the assay is fast, which alleviates the difficulties often associated with communicating the outcome of diagnostic tests. A prototype of the D4 assay is shown in Figure 3 below.
