Lab-on-a-Pill: wireless microfluidics for in vivo immunodiagnostics

Lead Research Organisation: University of Glasgow
Department Name: School of Engineering

Abstract

Previously, Lab-on-a-Chip technologies have exploited many aspects of microsystems technology, including both sensor miniaturisation and microfluidics, to produce technologies associated with DNA analysis, proteomics and diagnostics. Despite the numerous analytical advantages that are delivered as a consequence of miniaturisation into Lab-on-a-Chip, the vast majority of all devices that have been proposed (with the exception of handheld biosensors, first developed 20 years ago), most often require to be based on a laboratory bench. In contrast, wireless 'Lab-on-a-Pill' technology now has the proven ability to deliver both remote and-or distributed analysis, resulting in a wide range of potential applications, including those associated with biomedical analysis in the gastro-intestinal (GI) tract, process control in industry, environmental analysis and the functional foods industry. The concept is one of a battery-powered technology platform, which combines miniaturised sensors, a low power radio transmitter and receiver and power management modules, and an array of microsensors. Our current prototype can measure pH, disolved oxygen (pO2), temperature and conductivity, and has been proven with wireless measurements in a pig, using radiotelemtry to track its position. The device is controlled by an ASIC. This application now seeks to implement important technological challenges associated with implementing microfluidics on the Pill in order to enable advanced diagnostic tests. In this project we will illustrate this by performing remote immunodiagnostics. As an example, we will try to perform a remote biochemical assay for a marker for colon cancer in the lower GI tract.

Technical Summary

In this project, we propose, for the first time, to develop the technology to implement remote immunoassays in a Lab-on-a-Pill device. We propose to illustrate this generic technology by developing a specific application, namely that of an electrochemical assay for measuring blood in the GI tract (the electrochemical assay having the advantage that it can provide low power, high sensitivity measurements in a turbid environment). The work builds upon an established platform which has been developed in Glasgow University over the last four years. All functions will be under wireless control, including sample collection, microfluidic manipulation on the Pill (e.g. in providing the appropriate washing steps for a heterogeneous immunoassay), device calibration, and the transmission of results. The assay will be performed electrochemically, allowing for low power high sensitivity detection in a turbid and opaque sample. The assay will be configured as a heterogenous sandwich immunoassay for haemoglobin using an alkaline phosphatase labelled developing antibody, and 4 aminophenylphosphate as the substrate. Each assay will involve up to 5 individual fluidic manipulations on the pill, under wireless control. We have chosen to use a specialised packaging organisation, Optocap, to help enable the device to be produced as a workable package. The efficacy of the assay will be tested in pigs at St Marks Hospital.

Publications

10 25 50
 
Description The presence of blood in gastro-intestinal tract is a proxy for many clinically important conditions, not least bowel cancer,
where the detection of haemoglobin (Hb) in the faeces at concentrations of ca. 0.4 % may be disease marker (w/w,
equivalent to ca. 4 microg/mg of solid or 5 mg/ml in fluids in the colon).
The project had a number of clear outputs associated with this funding:


(i) The Development of a New Assay: 3,3',5,5'-Tetramethylbenzidine (TMB) was used as a redox mediator to explore the natural peroxidase activity of haemoglobin. The assay was performed as an immunoassay, where anti-human Hb (Abcam, UK) was immobilised on the surface of an Au-disc electrode using a standard chemistries based upon N
hydroxysuccinimide activation of a self-assembled monolayer of 11-mercaptoundecanoic acid (leading to reaction with free
amine groups on the surface of the antibody). Following a blocking step with a mixture of milk protein and BSA, to mitigate against non-specific adsorption of Hb, the immunosensor electrode was then immersed in a solution of Hb. After washing away unbound Hb, the bound-Hb antibody construct was then immersed in a solution containing TMB (2 mM), H2O2 (2 mM) and KCl (0.1 M) in phosphate-citrate buffer (pH 4.5). Current time transients were measured for various Hb concentrations between 0.0 and 8.0 mg mL-1. By configuring the assay so that we use the analyte (Hb) to generate the quantitative analytical response, we negate the need for secondary antibodies and enzyme labels, thus facilitating a 'label free' immunoassay.
This approach requires a much simpler assay protocol, which has fewer sample and reagent manipulation steps than for
traditional ELISA immunoassays. The work demonstrated the development of a new, sensitive, label-free electrochemical
immunoassay for Hb that utilises the intrinsic peroxidase activity of this clinically important analyte. The concentration
range for Hb measurement of this immunoassay accommodates the clincally relevant levels of Hb in fluids from the lower GI tract (ca. >0.5 mg mL-1). Importantly, our results also show that Hb retains its intrinsic peroxidase activity once bound by anti-Hb on the electrode surface. The assay not only provides a better biosensing route for determining Hb in turbid or opaque samples, when compared with its optical counter-part, but as a label free assay it requires fewer liquid handling steps (making it a promising candidate for successful miniaturisation onto a microfluidic platform). This formed the basis of a patent and a submitted publication.
(ii) Assay Formats: The assay was developed in three formats (a) Initially, the assay developed on standard macroscale
gold and screen printed electrodes (used for example by Mode DX in the in vitro testing units); (b) Considerable work was also performed in optimising the assay into a microsystems format, where the detection electrode was a gold or carbon paste microelectrode within a channel. This was developed in particular for use as a sensor within the Lab-Pill device; (c) The assay was also configured into a bead based assay, where the anti-Hb antibody was immobilised onto beads that increased the surface area. These beads "fished" analyte from a turbid sample (blood, faeces). Washing steps were performed by trapping the beads either physically or using a magnet.
(iii) System on Chip-Microsystems-software: As a central part of the project, an ASIC to control the Lab-Pill was designed.
More details of this are shown later. The chip was programmed and could control the sensors. The microsystems
technology (microfluidics) were controlled by a Labview programme, written in-house.
(iv) Methods: For handing blood, and faeces and combinations of the two, including human faeces and model matrix.
(v) Methods: Low power pumping methods were developed, based upon electrolysis of water.
Exploitation Route See previous report
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The formation of a company, ModeDx, which introduced a consumer bowel cancer detection system to the market place that was available through Boots, the Chemist.
First Year Of Impact 2014
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic