Elsevier

Biosensors and Bioelectronics

Volume 61, 15 November 2014, Pages 478-484
Biosensors and Bioelectronics

Autonomous capillary microfluidic system with embedded optics for improved troponin I cardiac biomarker detection

https://doi.org/10.1016/j.bios.2014.05.042Get rights and content

Highlights

  • Capillary microfluidics were fabricated for automated immunoassays.

  • Smart capillary microfluidics were designed comprising channels, pumps, trigger and delay valves.

  • We designed on-chip optical components to increase overall biosensing capabilities.

  • Biosensing was enhanced on-chip using multiple capture antibodies and molecular crosslinkers.

  • The system demonstrated a LOD of 24 pg/ml for troponin I cardiac biomarker.

Abstract

Cardiovascular diseases are the most prevalent medical conditions affecting the modern world, reducing the quality of life for those affected and causing an ever increasing burden on clinical resources. Cardiac biomarkers are crucial in the diagnosis and management of patient outcomes. In that respect, such proteins are desirable to be measured at the point of care, overcoming the shortcomings of current instrumentation. We present a CO2 laser engraving technique for the rapid prototyping of a polymeric autonomous capillary system with embedded on-chip planar lenses and biosensing elements, the first step towards a fully miniaturised and integrated cardiac biosensing platform. The system has been applied to the detection of cardiac Troponin I, the gold standard biomarker for the diagnosis of acute myocardial infarction. The devised lab-on-a-chip device was demonstrated to have 24 pg/ml limit of detection, which is well within the minimum threshold for clinically applicable concentrations. Assays were completed within approximately 7–9 min. Initial results suggest that, given the portability, low power consumption and high sensitivity of the device, this technology could be developed further into point of care instrumentation useful in the diagnosis of various forms of cardiovascular diseases.

Introduction

Immunosensing technologies have seen numerous applications in life sciences, medical diagnostics, environmental sampling and pharmaceutical analysis (Wild, 2005). With recent advances in the understanding of risk factors to human health, medical based immunsensing applications increasingly allow greater levels of patient care, improved diagnostics, and ultimately, a higher standard of living and management of critical diseases and conditions. Immunoassays are traditionally performed by centralised laboratories, however, immunodiagnostics at the point of care (POC) is slowly becoming a physical possibility (Chappel et al., 2009, Christenson and Azzazy, 2009, Warsinke, 2009).

Most successful POC based devices today include lateral flow based microfluidic functions, a well known commercialised example being the home pregnancy test kit. Such devices generally use an absorbent membrane that directs the flow of a sample to a test inspection area and which can also house all immunosensing reaction constituents. Either a colourimetric assay is performed providing a visible colour change, or a separate standalone reader provides post reaction quantification (Warsinke, 2009). Lab-on-a-chip technology has the potential to supersede the use of such lateral flow devices, through the use of lower reagents volumes, reduced overall footprint, accurate control of fluidic flow to match assay conditions and the ability to integrate more diverse surface chemistries and biosensing elements allowing thereby greater capacity for reaction quantification.

Laterally flow devices operate by means of capillary forces within the utilised paper and porous membranes, advantageously providing a means of fluidic flow with no energy input. Such forces can readily be created within microfluidics based systems through the interaction of a fluid with the wettable surface of a microchannel. Platforms have been demonstrated for precise controlled fluidic flow in microchannels (Clime et al., 2012, Hitzbleck and Delamarche, 2013, Man et al., 1998, Mohammed and Desmulliez, 2012, Mohammed and Desmulliez, 2013a, Mohammed and Desmulliez, 2014, Zimmermann et al., 2007, Zimmermann et al., 2008), containment of immunoassay reaction constituents on-chip (Gervais et al., 2011, Gervais and Delamarche, 2009), immobilisation of capture antibodies within the microchannels (Gervais et al., 2011, Gervais and Delamarche, 2009, Jonsson et al., 2008, Juncker et al., 2002, Mohammed and Desmulliez, 2013b) and reduction of the excitation and detection apparatus for portable endpoint fluorescence based quantification (Jonsson et al., 2008, Mohammed and Desmulliez, 2013b). Despite the impressive demonstrations of capillary immunosensing systems, to date platforms have been hindered by either the required use of large bench top imaging microscopes for the quantification of the reaction endpoints (Gervais et al., 2011, Gervais and Delamarche, 2009, Juncker et al., 2002), the lack of enhanced detection capabilities to maximise reaction/detection sensitivity (Jonsson et al., 2008, Juncker et al., 2002, Gervais et al., 2011, Gervais and Delamarche, 2009) or a lack of full reaction automation within the chip (Jonsson et al., 2008, Mohammed and Desmulliez, 2013b).

Of all the modern afflictions, cardiovascular related diseases are the most prevalent and life threatening medical conditions to affect the modern world (Mohammed and Desmulliez, 2011). Due to poor lifestyle choices and environmental factors amongst a growing world population, cases of such diseases are increasingly frequent across both high and low income countries and account for a significant drain on clinical resources. Detection of cardiac biomarkers is crucial for the diagnosis, triage and management of many serious cardiovascular related conditions, where time delay has a major impact on the short and long term health of a patient (Mohammed and Desmulliez, 2011). Despite the promise of microfluidic technology, very few POC cardiac biomarker immunosensing systems have been successfully commercialised, a possible exception being the Biosite Triage POC system (Clark et al., 2002). A recent systematic review of POC testing of cardiac biomarkers concluded that there is no ‘ideal test kit’ for the diagnosis of heart attacks, the most immediately life threatening cardiovascular condition, and existing tests were particularly criticised for the high number of reported false negatives, arguably making such devices less safe for current clinical implementation (Bruins Slot et al., 2013). Additionally, due to cost constraints and either lack of sensitivity or true portability, available commercial POC devices have failed to be fully integrated into routine clinical practise (Clark et al., 2002).

In response to these challenges, we demonstrate an automated biosensing platform with integrated optical components and enhanced biosensing elements for the rapid detection of the cardiac biomarker troponin I (cTnI), the current gold standard biomarker for the diagnosis of heart attacks. Such a system has for the first time been successfully developed using novel CO2 laser engraving to rapidly fabricate both advanced autonomous capillary microfluidics and planar lenses in a single manufacturing stage within a polymer substrate. The system is designed to be a self contained, cartridge style device, operating in conjunction with a portable, low power, fluorescence excitation and detection device to quantify the end point reaction on chip and in doing so, addressing the challenge of transferral of such technology from the laboratory and into ‘true’ portable use. Enhanced biosensing was achieved by the use of multiple detection and capture antibodies immobilised in a higher density and ‘ideal’ orientation through the use of molecular crosslinkers, in conjunction with the use of embedded microlenses. The device was tested using samples containing various serial dilutions of cTnI and was found to have a limit of detection (LOD) of 24 pg/ml, with a typical assay being completed within approximately 7–9 min.

Section snippets

Microchip materials and fabrication

Microfluidic devices were fabricated using a class 2, 10.6 μm CO2 laser etching system (Mini Helix 24, Epilog, UK). Designs were created using Corel Draw X4 (Corel Software, USA), which can be interfaced directly with the CO2 laser. Fabrication details using the CO2 ablation system were comprehensively reported previously by our group (Mohammed and Desmulliez, 2013a, Mohammed and Desmulliez, 2014). The complete chip was manufactured as a triple laminate layer, with microfluidic features

Autonomous flow

For ELISA type assays reaction reagents are typically added sequentially, with each phase requiring manual inputs by an operator, which can be highly laboured intensive and tedious when multiplied across several tens or hundreds of assays. When performed in a standard well plate format, an assay requires several hours to reach completion; however, the large surface to volume ratios in microfluidic systems will reduce this time frame down to the order of minutes due to improved reaction mixing

Conclusion

We have successfully demonstrated the manufacturing of advanced, autonomous capillary systems with embedded optical components and surface modification of PMMA substrate to allow for enhanced biosensing of the cTnI cardiac biomarker target. The device was fabricated entirely using a combination of rapid prototyping CO2 laser ablation and back-end processing techniques, allowing for the complete device to be fabricated rapidly, at low-cost and with high translatability for upscale manufacturing,

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