Find out how light-based chemical modification protocols of paper are paving the way for paper microfluidics and lateral flow assays.
Paper-based microfluidics and lateral flow assays are powerful platforms for fast and convenient diagnosis in point-of-care (PoC) formats. Besides simplicity these platforms of chemical modification promise savings for the healthcare system and applications in low-resource settings.
Dr Bastian E Rapp, of the NeptunLab at the Karlsruhe Institute of Technology (KIT), explains further.
Opportunities for lateral flow assays
Lateral flow assays can be carried out outside of a dedicated laboratory by personnel with low practical training, enabling analytics not only at the doctor’s office but also at a patient’s home. One of the best known lateral flow assays is the pregnancy test, highlighting the simplicity and widespread acceptance of these types of assays. Comparable tests have been established for numerous biomarkers where a positive binary readout (yes/no) is usually followed by a more in-depth laboratory test which elucidates the exact concentration of a biomarker.
This usually leads to significant reduction in cost because more expensive tests in centralised laboratories are only carried out when found necessary by a cost-effective and fast test. Lateral flow assays are discussed not only as an aid in everyday diagnosis but also for applications in low-resource settings and crisis scenarios whenever the available medical infrastructure is insufficient or fast sample-to-response intervals are required.
The current state-of-the-art in lateral flow assay is still binary readouts using relatively simple indicator reactions. However, many applications require quantitative information, i.e. the concentrations of a biomarker must be determined. Additionally, lateral flow assays are currently produced by adsorption protocols where the reagents required for a reaction are not covalently immobilised onto the paper and concentration gradients are difficult to manufacture.
However, concentration gradients are required if non-binary quantitative readouts are required. Antibodies (as required for ELISA-type assays) are often simply adsorbed on hydrophobic paper patches within a lateral flow assay, whereas the main substrate material used is usually hydrophilic to facilitate capillary flow. This implies that at least two (often even more) different types of papers have to be combined within the assay. Unspecific adsorption carries the additional risk of uncontrolled orientation and therefore degraded binding profiles and bioactivity of antibodies and similar biomolecules.
The aim of our approach for chemical modification of paper-based analytical devices and lateral flow assays was to develop a technique which allows:
- Manufacturing the assay from a single hydrophilic type of paper and
- The mild covalent immobilisation of biomolecules to the paper in a concentration-controlled manner, thereby facilitating concentration gradients and quantitative readouts.
In both cases, the technology is based on photochemistry and is thus accessible to photolithography, a well-established process in microengineering.
Hydrophobic barriers and chemical modification
Hydrophobic barriers to control the fluidic flow are generally introduced by either light-induced polymerising of a photoresist within the paper or impregnating the paper, for example with a wax applied by inkjet printing or transfer techniques. In both cases, the paper becomes stiff and brittle and thus susceptible to mechanical wear.
Our approach is based on a chemical modification of the paper structure itself. By a light-induced silanisation protocol, the paper’s wetting property is locally changed from hydrophilic to hydrophobic. This method retains the physical properties, for example, haptics, optical, and the mechanical properties of the paper. The silanisation is photoinduced and thus accessible to standard photolithography functionalising the paper only at the exposed areas. The mixture contains a hydrophobic alkoxysilane, a photoacid generator (PAG) and a photosensitiser.
The structure can either be defined with a mask or via a maskless projection lithography set-up (Waldbaur et al., 2012). The light activates the PAG, thereby hydrolysing the silane. The latter reacts with hydroxyl groups of the paper, rendering it hydrophobic. This results in a hydrophobic barrier which is robust even against harsh external influences such as kinking or strong bending.
Immobilisation of biomolecules
The second important pre-requisite for more complex chemical modification of paper-based analytical assays is a suitable technique for locally controlled immobilisation of biomolecules on the paper. For this, we developed a light-induced process based on photobleaching (Waldbaur et al., 2012) using cost-effective fluorescent dyes such as fluorescein, which bleaches quickly when exposed to light at a wavelength of 490nm. The photoradicals formed during the bleaching process allow covalent binding of the conjugated biomolecule to the surface of the paper.
For this, the paper must be pre-treated. We developed two methods for doing so. The first method is based on the adsorption of bovine serum albumin (BSA) to the paper. The adsorbed BSA serves as a radical acceptor for the photoradical, thus facilitating binding. The second method is based on the esterification of the hydroxyl groups of the cellulose. For this, the paper is treated overnight with methacrylic anhydride, resulting in a methacrylate surface. This non-saturated surface is able to react with the photoradical, forming a covalent bond. This technique allows binding a diverse group of biomolecules such as antibodies, streptavidin, and biotin to the surface of paper.
What was the conclusive result of chemical modification?
Lateral flow assays are an emerging platform for diagnostic applications. The two techniques developed by our lab and conducting chemical modification will allow the convenient, fast, and industry-compatible manufacturing of a wide range of analytical devices for applications in public health, low-resource settings, and clinical diagnostics.
- TM Nargang et al., ‘Structuring unbreakable hydrophobic barriers in paper, Microfluidics, BioMEMS, and Medical Microsystems XVI (2018)
- TM Nargang et al., ‘Functionalization of paper using photobleaching: a fast and convenient method for creating paper-based assays with colorimetric and fluorescent readout’, Engineering in Life Sciences (2016)
- E Wilhelm et al., ‘Protein assay structured on paper by using lithography,’ Microfluidics, BioMEMS, and Medical Microsystems XIII, 9320 (2015)
- A Waldbaur et al., ‘Maskless projection lithography for the fast and flexible generation of grayscale protein patterns’, Small, 8, 10, 1570-1578 (2012)
This work has been partly funded by the German Federal Ministry of Education and Research (BMBF), funding code: 03X5527 and 031A095C.