Controlled antibody release from gelatin for on-chip sample preparation

Abstract

A practical way to realize on-chip sample preparation for point-of-care diagnostics is to store the required reagents on a microfluidic device and release them in a controlled manner upon contact with the sample. For the development of such diagnostic devices, a fundamental understanding of the release kinetics of reagents from suitable materials in microfluidic chips is therefore essential. Here, we study the release kinetics of fluorophore-conjugated antibodies from (sub-) µm thick gelatin layers and several ways to control the release time. The observed antibody release is well-described by a diffusion model. Release times ranging from ~20 s to ~650 s were determined for layers with thicknesses (in the dry state) between 0.25 µm and 1.5 µm, corresponding to a diffusivity of 0.65 µm 2 /s (in the swollen state) for our standard layer preparation conditions. By modifying the preparation conditions, we can influence the properties of gelatin to realize faster or slower release. Faster drying at increased temperatures leads to shorter release times, whereas slower drying at increased humidity yields slower release. As expected in a diffusive process, the release time increases with the size of the antibody. Moreover, the ionic strength of the release medium has a significant impact on the release kinetics. Applying these findings to cell counting chambers with on-chip sample preparation, we can tune the release to control the antibody distribution after inflow of blood in order to achieve homogeneous cell staining. IntroductionOne of the major fields of applications for microfluidics is in vitro diagnostics, with great potential particularly in point-of-care diagnostics. 1-3 Many process steps and sensing principles have been realized on microfluidic devices. 4 Recently, on-chip sample preparation using reagents stored in microfluidic devices has received more and more attention. 5 On-chip sample preparation can eliminate the dependence on external instrumentation for reagent delivery as well as benchtop sample treatment, thus integrating the complete test into one disposable. 6 To realize on-chip sample preparation, reagents are integrated in microfluidic devices and released upon contact with the inflowing sample. Compared with liquid reagents 7-10 , integrating dry reagents in microfluidic devices is beneficial for long-term storage and convenient for transportation, due to the better stability of reagents in the dry state. 11 However, the controlled dissolution of the reagent and on-chip mixing with the added sample is challenging. Especially in applications where the inflowing sample passes a reservoir of the reagent and slowly dissolves or washes out the stored reagent 12-15 , very precise control of the sample flow and the reagent release process is required. In contrast, mixing between sample and reagents in stopped flow applications can be realized simply by releasing the reagent with sufficient delay after the sample inflow has stopped at the required position on the chip. 16 To realize controlled reagen...

Temperature-Switch Cytometry—Releasing Antibody on Demand from Inkjet-Printed Gelatin for On-Chip Immunostaining

“…Antibody release from gelatin layers was discovered to be strongly dependent on the conformation of the gelatin matrix . Freshly printed layers comprised small drops of gelatin, which are separately dispensed, and therefore dry so rapidly that mainly random entanglement between gelatin chains occurs and additional physical cross-linking via triple helix formation is suppressed. , Such rapidly dried gelatin will undergo continuous physical cross-linking via triple helix formation in a long-term structural evolution toward full maturation. , The increased degree of physical cross-linking in gelatin suggests a reduced mesh size in the gelatin matrix, thus causing an increased delay in antibody release. Obviously, optimal maturation conditions are essential to obtain the desired antibody release kinetics.…”

“…Chamber heights determined by interferometry were found to be 26.6 ± 0.4 μm ( n = 9). A more detailed description of the fabrication procedure can be found elsewhere …”

On-chip sample preparations for Point-of-Care cellular analysis of blood

“…This release delay is beneficial to prevent antibody wash-off in the chip (Figure 6i). By tuning the preparation of gelatine layers (thickness and degree of physical crosslinking), optimal release delay was achieved to maintain homogeneous antibody distribution in the chip, meanwhile ensuring more than 80 % antibody fractional release within 10 min for rapid and sufficient immunostaining of target cells [68] (Figure 6ii). The stained cells were imaged by a fluorescence analyser and the analysis of the obtained image yielded the cell count.…”

“…Layer maturation-induced delayed release and temperature-triggered release (lower). i) copyright 2012 Royal Society of Chemistry [67]; ii) copyright 2016 Royal Society of Chemistry [68]; iii) [69].…”

Hands-free cytometry of whole blood : controlled antibody release from hydrogels for on-chip cell staining

“…1.6i) By tuning the preparation of gelatin layers (thickness and degree of physical crosslinking), optimal release delay was achieved to maintain homogeneous antibody distribution in the chip, meanwhile ensuring more than 80% antibody fractional release within 10 min for rapid and sufficient immunostaining of target cells. 71 ( Fig. 1.6ii) The stained cells are imaged and the analysis of the acquired image yields the cell count.…”