Highly sensitive immunoassay based on controlled rehydration of patterned reagents in a 2-dimensional paper network.

We have demonstrated a multistep 2-dimensional paper network immunoassay based on controlled rehydration of patterned, dried reagents. Previous work has shown that signal enhancement improves the limit of detection in 2-dimensional paper network assays, but until now, reagents have only been included as wet or dried in separate conjugate pads placed at the upstream end of the assay device. Wet reagents are not ideal for point-of-care because they must be refrigerated and typically limit automation and require more user steps. Conjugate pads allow drying but do not offer any control of the reagent distribution upon rehydration and can be a source of error when pads do not contact the assay membrane uniformly. Furthermore, each reagent is dried on a separate pad, increasing the fabrication complexity when implementing multistep assays that require several different reagents. Conversely, our novel method allows for consistent, controlled rehydration from patterned reagent storage depots directly within the paper membrane. In this assay demonstration, four separate reagents were patterned in different regions of the assay device: a gold-antibody conjugate used for antigen detection and three different signal enhancement components that must not be mixed until immediately before use. To show the viability of patterning and drying reagents directly onto a paper device for dry reagent storage and subsequent controlled release, we tested this device with the malaria antigen Plasmodium falciparum histidine-rich protein 2 (PfHRP2) as an example of target analyte. In this demonstration, the signal enhancement step increases the visible signal by roughly 3-fold and decreases the analytical limit of detection by 2.75-fold.

Controlled release of dry reagents in porous media for tunable temporal and spatial distribution upon rehydration.

Novel methods are demonstrated that enable controlled spatial and temporal rehydration of dried reagents in a porous matrix. These methods can be used in paper-based microfluidic assays to define reagent concentrations over time at zones downstream for improved performance, and can reduce costs by simplifying the manufacturing process with the use of a single porous substrate. First, the creation of uniform reagent pulses from patterned arrays of dried reagent is demonstrated. Second, reagents are stored dry in separate regions of the porous matrix so that they can be combined upon rehydration for immediate use in the device. Third, reagents are reconstituted sequentially from dry storage depots with tunable delivery times. Fourth, the total time for dissolution is varied to achieve a range of reagent delivery times to a downstream region. Finally, the utility of these control methods is demonstrated in the context of real-time reagent rehydration and mixing on a porous device.

Multiplexed inkjet functionalization of silicon photonic biosensors.

The transformative potential of silicon photonics for chip-scale biosensing is limited primarily by the inability to selectively functionalize and exploit the extraordinary density of integrated optical devices on this platform. Silicon biosensors, such as the microring resonator, can be routinely fabricated to occupy a footprint of less than 50 × 50 µm; however, chemically addressing individual devices has proven to be a significant challenge due to their small size and alignment requirements. Herein, we describe a non-contact piezoelectric (inkjet) method for the rapid and efficient printing of bioactive proteins, glycoproteins and neoglycoconjugates onto a high-density silicon microring resonator biosensor array. This approach demonstrates the scalable fabrication of multiplexed silicon photonic biosensors for lab-on-a-chip applications, and is further applicable to the functionalization of any semiconductor-based biosensor chip.