Passive fluidic chip composed of integrated vertical capillary tubes developed for on-site SPR immunoassay analysis targeting real samples

Abstract

We have successfully developed a surface plasmon resonance (SPR) measurement system for the on-site immunoassay of real samples. The system is composed of a portable SPR instrument (290 mm(W) × 160 mm(D) × 120 mm(H)) and a microfluidic immunoassay chip (16 mm(W) × 16 mm(D) × 4 mm(H)) that needs no external pump system. An integrated vertical capillary tube functions as a large volume (150 μL) passive pump and a waste reservoir that has sufficient capacity for several refill operations. An immunoassay was carried out that employed the direct injection of a buffer and a test sample in sequence into a microfluidic chip that included 9 antibody bands and 10 reference reagent bands immobilized in the flow channel. By subtracting a reliable averaged reference sensorgram from the antibody, we effectively reduced the influence of the non-specific binding, and then our chip successfully detected the specific binding of spiked IgG in non-homogeneous milk. IgG is a model antigen that is certain not to be present in non-homogeneous milk, and non-homogeneous milk is a model of real sample that includes many interfering foreign substances that induce non-specific binding. The direct injection of a real sample with no pretreatment enabled us to complete the entire immunoassay in several minutes. This ease of operation and short measuring time are acceptable for on-site agricultural, environmental and medical testing.

Keywords: SPR; immunoassay; microfluidics; on-site; passive pump; raw sample.

Figure 1.

Structure of microfluidic chip for SPR measurement composed of integrated vertical capillary enclosure made of clear acrylic resin (A), thin plastic flow channel film (B), gold/titanium sputtered substrate with immobilized antibodies and enzyme in band array configuration (C), and photograph of finished product (D). The chip is mounted on a portable SPR instrument and sample liquid is injected with pipette (E). Schematic diagram of the portable SPR instrument, (F). Optical configuration at the interface of the instrument and the sensor chip, (G).

Figure 2.

Flow rate distributions of passive fluidic chips calculated from the run-off time of 10 μL injections into the inlet. The run-off time was measured by observing the bottom of the inlet.

Figure 3.

Photograph of spotted antibodies and enzyme in a band array structure (center), their abbreviations (right) and the SPR signal at the center of each band (left). The blocking reagent (Block Ace) was spotted on both sides of the antibodies and enzyme as a reference in difference sensorgrams.

Figure 4.

Immunoassay analysis of spiked IgG in non-homogenized milk using a portable SPR instrument and a microfluidic chip. The selected raw sensorgrams A and their difference sensorgrams B. The selected sensorgrams are measured at the specific antibody of the target antigen: anti human IgG (12:I3382), the non-specific antibody of the target antigen: anti Staphylococcal alpha Hemolysin (14:S5V156-754), and their references on both sides. Each difference sensorgram is obtained by subtracting an average reference sensorgram from that of the antibody. An average reference sensorgram is the average of reference sensorgrams located on either side of the antigen.

Figure 5.

Calibration curve of the SPR immunoassay measured with a spiked antigen in non-homogenized milk. The relationship between the antigen concentration and the slope of the sensorgrams. Specific antigen-antibody pair 12 (squares) and non-specific pairs 10 (triangles) and 14 (diamonds) are shown in the same graph. A small offset in the horizontal axis was used for non-specific pairs to give clear view of the error bar overlaps. The solid line in the graph is the result of linear regression analysis applied to the specific pair.