Preparation of Linear Cryogel Arrays as a Microfluidic Platform for

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Preparation of Linear Cryogel Arrays as a Microfluidic Platform for Immunochromatographic Assays Marc Zinggeler, Patrick Fosso, Yan Hao, Thomas Brandstetter, and Jürgen Rühe Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 23, 2017

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Analytical Chemistry

Preparation of Linear Cryogel Arrays as a Microfluidic Platform for Immunochromatographic Assays Marc Zinggeler, Patrick L. Fosso, Yan Hao, Thomas Brandstetter and Jürgen Rühe* Laboratory for Chemistry and Physics of Interfaces, Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany, E-mail: [email protected] ABSTRACT We describe a new microfluidic platform to perform immunochromatographic assays. The platform consists of a linear assembly of small, porous cryogel monoliths functionalized with different biomolecules and anchored in an optically transparent capillary, which serves as the microfluidic carrier. This assembly enables fluid flow by capillary action and simple optical detection. Using an in-situ preparation method, individual compartments are generated from small plugs of polymer solutions that are subsequently transformed into individually functionalized cryogel monoliths through a photo induced crosslinking reaction. In the same reaction step the monoliths are firmly anchored to the surface of the capillary. A proof-of-concept, prototype platform is successfully used for the detection of the inflammatory marker interleukin 6 via a sandwich immunoassay. We observe excellent assay performance metrics that includes high sensitivity, good linearity and low variation. We also demonstrate fluid transport solely by passive means which is a critical attribute for point-of-care diagnostics. Immunochromatographic assays like the lateral flow assay (LFA) are highly suited for the analysis of various biomarkers in resource-limited point-of-care (POC) settings.1–4 At the heart of a typical LFA lies a modified nitrocellulose membrane, which enables probe flow by 1 ACS Paragon Plus Environment


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capillary action, freeing it from external equipment and enabling one-step binding assays by providing all required assay components along the test stripe. Due to its handling simplicity, cost-effectiveness, robustness, high market presence and number of implemented lab-on-achip applications, the LFA may be regarded as a gold standard in the field of microfluidics,5 which has paved the way to the recent revival of paper-based microfluidic devices.6–10 Nevertheless, there are many cases where the performance of LFAs is still insufficient. One of the principal weaknesses that has been identified is the non-specific interactions of assay and sample components with the hydrophobic nitrocellulose membrane.2,11 Though, this problem can be reduced by applying blocking agents like detergents or polymers to the membrane, these agents can also critically interfere with the assay.2,11 Other identified weaknesses include limited options for the chemical immobilization of biomolecules (which is restricted to passive adsorption)2,12 and the unfavorable optical properties, which limit the achievable sensitivity during optical detection.6,8,13 To address these limitations, we propose a new platform for immunochromatographic assays that consists of a linear assembly of porous hydrophilic polymer monoliths. The monoliths are composed of cryogels generated inside a capillary, which acts as a transparent microfluidic carrier and enables fluid transport by capillary action. The different monoliths are individually functionalized by immobilization of chosen biomolecules such that the system is in this respect analogous to a classical LFA. Polymeric cryogels are chosen to form the monoliths as they possess a unique porous structure, high mechanical stability and hydrophilic nature.14 Cryogels are macroporous hydrogels prepared by crosslinking of monomeric or polymeric precursor solutions in the frozen state (also known as cryogelation). Freezing induces a microphase separation between the crystallized solvent (typically water) and an unfrozen fraction of concentrated precursor solution called the “liquid microphase” (LMP). After

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crosslinking, which only occurs within the LMP, the solvent crystals are thawed to obtain a network of interconnected pores.15 The problem with the presently known in-situ preparation methods,16–19 which means that the monoliths are directly synthesized in the system itself, is that most polymerization or crosslinking strategies are not compatible with the presence of biomolecules. This problem could be solved by following a step-by-step approach in which additional immobilization reactions are performed after monolith formation. For this, however, one must resort to ex-situ processes, as an individual functionalization of the different compartments would otherwise be extremely challenging. Using an ex-situ method, where the different monoliths are first synthesized and functionalized outside the device before incorporation,20,21 however, would be more complex and would only possess a strongly limited potential in respect to automatization, miniaturization and multiplexing. To overcome this technical hurdle, we developed an in-situ method for the preparation of linear arrays of individually functionalized cryogel monoliths (see Figure 1). The method is based on a cryogelation method we recently introduced that enables the preparation of biofunctional cryogels within a single photo-reaction step.22 The process begins with the preparation of aqueous solutions consisting of a copolymer (poly (N,N-dimethylacrylamidestat-methacryloyloxy benzophenone-stat-sodium-4-styrenesulfonate))23 containing photoactive benzophenone units and the biomolecules to be immobilized. Known small volumes of these solutions are injected, one after the other, into a suitable transparent carrier (e.g. a plastic or glass capillary) by applying controlled low pressure. Different solutions are separated from each other using air pockets to space liquid injections. Subsequently, all solutions are frozen to obtain phase-separation between the growing ice crystals and the LMPs. After freezing, the system is illuminated with UV-light (365 nm) to induce the photochemical reactions in the LMPs. The UV-light excites the benzophenone units contained 3 ACS Paragon Plus Environment


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in the photopolymer into a biradicaloid triplet state that can readily abstract hydrogen from proximal C-H groups. Radical recombination leads to formation of covalent C-C bonds.24 Because the copolymer, the plastic substrate and the biomolecules to be immobilized, all possess an abundance of accessible C-H groups this process causes simultaneous crosslinking of the copolymer chains, biomolecule immobilization and surface attachment all at once. To obtain a stable attachment of the forming cryogels to glass substrates, a pretreatment of the glass surface with a suitable organosilane (alkylsilane or benzophenone silane) is recommended, plastic capillaries can be used as obtained. After the photochemical reactions have been completed, the assembly is allowed to thaw to obtain stable cryogel arrays. Due to the simple handling of the liquid compartments and its collective transformation to completed monoliths within a single reaction step, the process is considered to have high automatization, miniaturization and multiplexing potential. Additionally, the universal reactivity of the benzophenone chemistry renders the process highly versatile and enables a broad choice of carriers and functionalities.

Figure 1. Schematic depiction of the developed process for the preparation of linear cryogel arrays in a transparent carrier. 4 ACS Paragon Plus Environment

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The utility of the new platform was demonstrated at the example of an assay for the detection of the plasma cytokine interleukin 6 (IL-6), which is one of the most important inflammatory cytokines.25 Cytokines can be considered as the most challenging class of plasma protein biomarkers in respect of device sensitivity due to their ultra-low concentration in the range of pg/ml.26 In the case of IL-6 the blood buffer of healthy individuals is in the range of 1 pg/ml and can raise up to the low µg/ml range in the case of a septic shock.25 However, there is a strong clinical interest in detecting already slightly elevated IL-6 levels to guide the early assessment of sepsis.27 The performances of commercial ELISA and LFA platforms for the analysis of IL-6 were recently compared and sensitivity values of 0.99) with low variation and a high sensitivity. The sensitivity values are based on the fitted calibration curves and indicate a limit of detection (LOD) of 26 pg/ml in buffer and 30 pg/ml in plasma (LOD-signal = blank + 3σ). These results confirm the high compatibility of the cryogel monoliths with biological samples and demonstrate the feasibility of highly sensitive diagnostic assays performed in the developed device. The LOD of the described system for the detection of IL-6 is lower than the reported value of 50 pg/ml of a commercial POC device28 and is more than adequate for most other protein biomarkers. This indicates that the incubation time of 90 min, which was used in the presented case, could significantly be reduced for most applications, paving the way for 7 ACS Paragon Plus Environment


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the realization of rapid tests which are typically read-out before thermodynamic equilibrium is obtained. On the other hand, the sensitivity of the system could further be enhanced by increasing the load of capture antibodies and applying signal amplification strategies, which are widely used to boost the performance of classical LFAs.30

Figure 3. Schematic depiction and analytical results of the IL-6 demonstrator platform. a) Schematic of the performed sandwich immunoassay for the analysis of interleukin 6 (IL-6). b) White light (top) and fluorescence (middle and bottom) images of capillaries showing typical assay results. c) Measured net signals (= T – NC) of capillaries with different IL-6 incubation times (n = 3, mean values and standard deviations are shown) for an IL-6 concentration of 500 pg/ml in buffer. d) Measured net signals for different IL-6 concentrations (90 min incubation) measured in buffer (black dots) and non-diluted blood plasma (grey triangles) and fitted calibration curves (solid and dashed line in respective colors).

The approach which was used to perform this first assays in the laboratory setting involved a pump and a bench-top fluorescence reader and is therefore not directly suited for a POC application. As already discussed, one key strength of LFAs is the passive fluid transport, which is also considered an important prerequisite for the successful realization of POC compatible, rapid tests based on linear cryogel arrays. To demonstrate the feasibility of this 8 ACS Paragon Plus Environment

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concept, one of the prepared IL-6 prototypes was first dried and connected to a commercial absorbent material (wick) on one end, before it was brought in contact with a droplet (50 µl) of black ink on the other end (see Figure 4). The ink allows a simple visual inspection of the filling process. In this configuration upon contact with the liquid, the device was successfully filled by the capillary action of the carrier followed by continued fluid transport accomplished by the hydration of the wick. The integrity of the device was not affected by the drying and re-hydration procedure, rendering it suitable for storage in the dry state, which is preferred for POC applications. Currently, we are further developing the platform in this direction, involving the integration of all required assay components in the capillary to realize one-step binding assays and using a hand-held device for fluorescence detection.

Figure 4. Image sequence showing passive fluid transport through a glass capillary containing three cryogel monoliths connected to an absorbent material (wick).

CONCLUSIONS Linear cryogel arrays represent a novel platform for immunochromatographic assays. To realize such a platform, a simple and versatile in-situ method for the preparation of linear cryogel arrays based on photochemically induced C,H-insertion crosslinking reactions in transparent carriers was developed. The proposed approach allows a high degree of 9 ACS Paragon Plus Environment


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automatization of the device preparation and a simple miniaturization as well as multiplexing of the thus generated assays. The individual cryogel monoliths described here had volumes of about 450 nl. The generation of much smaller gels is easily doable, however, might make the optical detection unnecessarily complicated. Using this method a prototype platform for the analysis of IL-6 based on a sandwich immunoassay was successfully realized. The platform showed excellent assay performance in first proof-of-concept experiments, which were performed by dynamic incubation through active pumping. However, such devices can also be performed using passive fluid transport, which was successfully demonstrated in an exemplary case. Passive fluid transport through the device is considered an important prerequisite for the further development of the system towards an application in POCdiagnostics.

ACKNOWLEDGEMENTS The authors acknowledge Prof. Dr. William J. Brittain for proof-reading the manuscript. This research was supported by the Bundesministerium für Bildung und Forschung - BMBF (FKZ031B0213D).

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Preparation of Linear Cryogel Arrays as a Microfluidic Platform for

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