Ioannis Katis

Paper-based colorimetric enzyme linked immunosorbent assay fabricated by laser induced forward transfer

We report the Laser Induced Forward Transfer (LIFT) of antibodies from a liquid donor film onto paper receivers for application as point-of-care diagnostic sensors. To minimise the loss of functionality of the active biomolecules during transfer, a dynamic release layer was employed to shield the biomaterial from direct exposure to the pulsed laser source. Cellulose paper was chosen as the ideal receiver because of its inherent bio-compatibility, liquid transport properties, wide availability and low cost, all of which make it an efficient and suitable platform for point-of-care diagnostic sensors. Both enzyme-tagged and untagged IgG antibodies were LIFT-printed and their functionality was confirmed via a colorimetric enzyme-linked immunosorbent assay. Localisation of the printed antibodies was exhibited, which can allow the creation of complex 2-d patterns such as QR codes or letters for use in a final working device. Finally, a calibration curve was determined that related the intensity of the colour obtained to the concentration of active antibodies to enable quantitative assessment of the device performance. The motivation for this work was to implement a laser-based procedure for manufacturing low-cost, point-of-care diagnostic devices on paper.

Laser-based patterning for fluidic devices in nitrocellulose

In this report, we demonstrate a simple and low cost method that can be reproducibly used for fabrication of microfluidic devices in nitrocellulose. The fluidic patterns are created via a laser-based direct-write technique that induces polymerisation of a photo-polymer previously impregnated in the nitrocellulose. The resulting structures form hydrophobic barriers that extend through the thickness of the nitrocellulose and define an interconnected hydrophilic fluidic-flow pattern. Our experimental results show that using this method it is possible to achieve microfluidic channels with lateral dimensions of ∼100 μm using hydrophobic barriers that form the channel walls with dimensions of ∼60 μm; both of these values are considerably smaller than those that can be achieved with other current techniques used in the fabrication of nitrocellulose-based fluidic devices. A simple grid patterned nitrocellulose device was then used for the detection of C-reactive protein via a sandwich enzyme-linked immunosorbent assay, which served as a useful proof-of-principle experiment.

Laser direct-write for fabrication of three-dimensional paper-based devices

We report the use of a laser-based direct-write (LDW) technique that allows the design and fabrication of three-dimensional (3D) structures within a paper substrate that enables implementation of multi-step analytical assays via a 3D protocol. The technique is based on laser-induced photo-polymerisation, and through adjustment of the laser writing parameters such as the laser power and scan speed we can control the depths of hydrophobic barriers that are formed within a substrate which, when carefully designed and integrated, produce 3D flow paths. So far, we have successfully used this depth-variable patterning protocol for stacking and sealing of multi-layer substrates, for assembly of backing layers for two-dimensional (2D) lateral flow devices and finally for fabrication of 3D devices. Since the 3D flow paths can also be formed via a single laser-writing process by controlling the patterning parameters, this is a distinct improvement over other methods that require multiple complicated and repetitive assembly procedures. This technique is therefore suitable for cheap, rapid and large-scale fabrication of 3D paper-based microfluidic devices.

Engineering fluidic delays in paper-based devices using laser direct-writing

We report the use of a new laser-based direct-write technique that allows programmable and timed fluid delivery in channels within a paper substrate which enables implementation of multi-step analytical assays. The technique is based on laser-induced photo-polymerisation, and through adjustment of the laser writing parameters such as the laser power and scan speed we can control the depth and/or the porosity of hydrophobic barriers which, when fabricated in the fluid path, produce controllable fluid delay. We have patterned these flow delaying barriers at pre-defined locations in the fluidic channels using either a continuous wave laser at 405 nm, or a pulsed laser operating at 266 nm. Using this delay patterning protocol we generated flow delays spanning from a few minutes to over half an hour. Since the channels and flow delay barriers can be written via a common laser-writing process, this is a distinct improvement over other methods that require specialist operating environments, or custom-designed equipment. This technique can therefore be used for rapid fabrication of paper-based microfluidic devices that can perform single or multistep analytical assays.

Improved sensitivity and limit-of-detection of lateral flow devices using spatial constrictions of the flow-path

We report on the use of a laser-direct write (LDW) technique that allows the fabrication of lateral flow devices with enhanced sensitivity and limit of detection. This manufacturing technique comprises the dispensing of a liquid photopolymer at specific regions of a nitrocellulose membrane and its subsequent photopolymerisation to create impermeable walls inside the volume of the membrane. These polymerised structures are intentionally designed to create fluidic channels which are constricted over a specific length that spans the test zone within which the sample interacts with pre-deposited reagents. Experiments were conducted to show how these constrictions alter the fluid flow rate and the test zone area within the constricted channel geometries. The slower flow rate and smaller test zone area result in the increased sensitivity and lowered limit of detection for these devices. We have quantified these via the improved performance of a C-Reactive Protein (CRP) sandwich assay on our lateral flow devices with constricted flow paths which demonstrate an improvement in its sensitivity by 62x and in its limit of detection by 30x when compared to a standard lateral flow CRP device.

Rapid Multiplexed Detection on Lateral-Flow Devices Using a Laser Direct-Write Technique

Paper-based lateral flow devices (LFDs) are regarded as ideal low-cost diagnostic solutions for point-of-care (POC) scenarios that allow rapid detection of a single analyte within a fluidic sample, and have been in common use for a decade. In recent years, there has been an increasing need for rapid and simultaneous detection of multiple analytes present within a single sample and to facilitate this, we report here a novel solution—detection using a multi-path LFD created via the precise partitioning of the single flow-path of a standard LFD using our previously reported laser direct-write (LDW) technique. The multiple flow-paths allow the simultaneous detection of the different analytes individually within each of the parallel channels without any cross-reactivity. The appearance of coloured test lines in individual channels indicates the presence of the different analytes within a sample. We successfully present the use of a LDW-patterned multi-path LFD for multiplexed detection of a biomarker panel comprising C-reactive protein (CRP) and Serum amyloid A-1 (SAA1), used for the diagnosis of bacterial infections. Overall, we demonstrate the use of our LDW technique in the creation of a novel LFD that enables multiplexed detection of two inflammation markers within a single LFD providing a detection protocol that is comparatively more efficient than the standard sequential multiplexing procedure.

Laser engineering of three-dimensional (3D) structures in paper-based microfluidic devices

Microfluidic engineering technology has been widely used for implementing lab-on-chip (LOC) type point of care (POC) devices since its origins in the 1990s. LOC devices offer some distinct advantages such as the possibility of reducing the quantity of valuable samples or reagents needed and the shortening of the detection times, both of which result from their compact structure [1]. Paper-based microfluidic devices, which are regarded as a low-cost alternative to conventional POC diagnostics tools, have also been popularly studied in the last few decades, and a huge number of advantages have been explored, such as low-cost, mass production, disposability, being equipment free etc. [2] However, some disadvantages or limitations are also apparent, such as issues with the control of flow rate, multiplexed detection on the same device footprint and further reduction of size [2]. As a result, 3D microfluidic paper analytical devices, as an alternative solution, have been proposed in recent years, which enable distribution of fluids in both lateral and vertical directions.

Laser-direct-write methods for fabrication of paper-based medical diagnostic sensors

We demonstrate the use of laser-based direct-write methods, namely laser-induced forward transfer and laser-induced photo-polymerization as printing and patterning tools for the fabrication of paper-based fluidic sensors that enable affordable point-of-care medical diagnostics.

Rapid, low-cost patterning of microstructures in polydimethylsiloxane via mask-less laser-machining

Summary form only given. Polydimethylsiloxane (PDMS), due to its unique characteristics including biocompatibility, possibility to be flexibly moulded into the desired shape, optical transparency, and its low-cost provides a valuable advantage as a building material for the fabrication of microfluidics-based lab-on-chip devices and micro-contact printing [1, 2] moulds that allow parallel deposition of various materials on a target surface. A range of methodologies such as wet chemical etching, dry plasma etching, decal transfer microlithography, and bond-detach method have been utilised for creating patterns in PDMS. However, one of the most commonly used approaches for the prototyping of PDMS for such applications is soft-lithography. This involves the use of a clean-room based UV-lithography step that uses expensive custom-designed masks for the fabrication of a master-mould with structures that are then duplicated via stamping of this master to produce a secondary-mould in PDMS, which is then used for micro-contact printing applications. Even though this lithographic procedure can routinely produce high-resolution micron-scale structures, the procedure is time-consuming and expensive. Instead, for the production of the master, we propose as a cheap alternative to expensive UV-lithography, a mask-less laser-based procedure which does not rely on cleanroom access. Similar to the soft-lithographic procedure, the process is two-step and allows the creation of high-quality sub-micron to millimetre-scale features, in a wide range of materials, with the added advantage of being able to fabricate complex and differently-shaped structures adjacent to each other, in either a sequential or a single-step. This laser-based method (Fig.1) has been used to create two-dimensional surface relief patterns in a master-mould for replication into PDMS and subsequent contact-printing.