Sample preparation description

Miniaturisation of biological assays has very well known advantages. However, it has a simple disadvantage: the sample to be introduced in the LOC is also miniaturised. Consequently, the smaller the sample, the smaller the chance to detect the target, and in turn, the smaller the signal. For example, to detect Campylobacter in chicken by DNA methods, it is crucial to take at least one bacteria in the sample volume. It is essential to include within the sample preparation to carry out a concentration step of raw sample (food, blood, urine, faeces or biopsy cells). Despite high miniaturisation of some preparation steps, the MEMS community has not integrated all functionalities to allow raw sample analysis within a LOC. The most remarkable works are dealing with magnetophoresis concentrators. There is a clear synergy between Magnetic Beads and LOC due to the fact that thin walls of the fluidic chambers make very efficient magnetic beads trap. Some miniaturisations of these operations have been achieved: PCR thermocycling, cell separation, denaturation of DNA, PCR optical detection, and cell lysis.

More recently, two different works have been published and patented dealing with sample preparation and PCR: a work from Samnsung [7] and a work from partners within the proposal consortium (work carried out in the framework of OPTOLABCARD European project FP6-STREP-016727). Both devices are capable of concentrate target microorganisms, lyse them and amplify the extracted DNA by real time-PCR (qPCR) in a single microchamber [14,15]. However the work belonging to the consortium shows a better performance: (i) real human samples were used; (ii) microorganisms concentration, lysis and qPCR were carried out in a single microchamber; (iii) less PCR mixture (2.5 μl) was required; (iv) less time (25 minutes) was needed for the complete analysis and (v) a higher sensitivity (CT=15) was obtained.

Nevertheless, these two works still have limitations:

  • Fabrication based on wafers increases the price of the LOC.
  • Limited sample preparation by using just one chamber (impossible to carry out washing steps, reagents are not stored within the LOC, valves not integrated).
  • Complex optical reader.
  • Only DNA detection.
  • Devices limited to rigid substrates (no SmartCards and no Skin Patches).

A handheld unit performing NASBA with real-time fluorescence amplification monitoring has been developed by the University of Southern Florida (USF) and used (e.g. for the measurement of rbcL gene of K. brevis (a ‘red tide’ HAB (Harmful Algae Bloom) species)). This device has been used extensively for analysis of RNA for a number of phytoplankton species and communities, for Enteroviruses and Noroviruses. An in situ (i.e. autonomous and submersible) oceanographic NASBA analyser has been developed based on this technology.

This shows the potential of the device proposed in this application, which extends the concept to a more compact device, that can perform multiple analysis (qPCR and qIPCR in addition to NASBA) and can be rapidly reconfigured for each analysis (by use of the card / reader system).

An alternative approach using immobilised hybridization probes has been developed to produce an in situ instrument for sample preparation, lysis and detection of phytoplankton (the ‘ESP’ instrument. This approach gives a semi-quantitative indication of species presence in the sample and has the advantage of multiplexed read out. This achieves a microbiologists first goal: identifying who is present.

The device proposed in this application, with multiple analyses available, will also determine how many of each genotype is present, what they are doing, and what is their physiological state. This gives a near complete picture of the microbial environment.

Immobilized hybridization probes are also developed for a wide range of marine species under European grant "GOCE-CT-2003-505491". This lab based system produces results to the ‘ESP’ instrument described above, but with a greater degree of multiplexing and a wider range of target species. Handheld electrochemical biosensors based on PCR have been developed. These have been applied to HAB detection and can detect ~103 cells/l in 3-5hours.

By comparison the proposed device offers better detection levels, a wider range of analysis, and faster reconfiguration.

The right balance between relevant, feasible and efficient research.
IKERLAN-IK4, Technological Research Centre. | J.M. Arizmendiarrieta N2 | 20500 Arrasate-Mondragon | Spain | jmruano@ikerlan.es