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Sound Thinking: A Disruptive Rethink of In Vitro Diagnostics
Diagnostic testing and how this has been conducted to date
Diagnostic testing is now performed in a wide range of settings: hospital laboratories, independent laboratories (including large reference laboratories), primary care facilities and, in the case of tests such as pregnancy tests and blood glucose tests, even at home. In general, these tests require a number of components, including an instrument and reagents, often packaged in disposable cartridges.
Tests can be performed on samples of blood, urine, stool, tissue, or via a swab. Some are simple and can be performed reliably at the point of care (POC) in a doctor’s surgery, clinic or at home. Others require expensive equipment and supplies, sophisticated methods, skilled technicians, and specialised personnel to interpret results. These are usually performed in large laboratories.
PCR: The gold-standard in diagnostics
Molecular diagnostics revolves primarily around the Nucleic Acid Test (NAT). This is used to detect genomic DNA or RNA sequences.
One key molecular diagnostic method that underpins many of these tests is the Polymerase Chain Reaction (PCR). This technique has many diverse applications: in infectious disease diagnostics for detection of viruses or bacteria; in forensic science; paternity tests; security applications; and myriad commercial applications.
The invention of PCR in 1983 was one of the many great medical advances of the last century.
Over the years, many variants of the original system have been developed, perhaps the most important being the Real-Time PCR Analysis System. This enables the amplification, monitoring, and quantification of the number of DNA copies in the sample under consideration, even if the starting amount of material is at a very low concentration.
It is commonly referred to as quantitative PCR (qPCR) and eliminates the need for post-processing to measure the PCR product. However, such tests require well-equipped laboratories with sample preparation apparatus, such as centrifuges and thermal cyclers.
Big machines. Low portability. High costs.
Real-time PCR systems are complex, comprising a heater, temperature sensors and detection units. Commercial units are still typically cabinet-sized boxes designed for high-throughput mass screenings.
The temperature cycling, that underpins the whole process calls for many cycles of heating and cooling of samples, with thermoelectric coolers used to speed up the whole process. Such systems usually require significant power and are generally impractical for testing away from the laboratory.
Smaller, but just as many problems
POC applications, in which the test is taken to the patient, require portability and low power consumption.
Such smaller, ‘Lab-on-a-Chip’ devices, often based on microfluidics, have entered the market over the years, using either ‘time-domain’ or spatial-domain strategies to move samples between heaters, and enabling a degree of automation. Perhaps the most interesting of these are so called ‘Sample-to-Answer’ machines. The Cepheid® GeneXpert® system, introduced in 2004, led the way for this kind of device.
Using small disposable cartridges, barcoded and pre-loaded with reagents for particular assays, this system is able to address raw sample processing, real-time PCR, and fluorescent monitoring of multiple channels for target and control signals. The software then interprets these, and in an hour or so from sample addition, with no intervention by a technician, a results call is generated. A range of processor sizes are available to handle different numbers of random-access cartridges at any given time, and a wide range of assays is also available.
Like their larger forebears, Sample-to-Answer devices of this kind still have downsides. At the forefront of these is costs, both per instrument and per test (each of which uses a consumable cartridge). Costs are usually appreciably higher than running the equivalent molecular test in a traditional assay system and can be a prohibitive downside where large sample throughputs are called for.
Furthermore, the machines require daily, weekly, monthly, and yearly maintenance with regular service tasks which are extensive and complex.
Modern devices still using traditional diagnostics
Despite the downsides of small-form devices still essentially executing the same process - albeit miniaturised - as traditional laboratory devices, it is important to recognise the benefits of instruments of this kind.
They can be operated by staff with far less specialised training than is needed for a traditional molecular lab technologist.
They bypass the need for many of the specialised infrastructural requirements of a traditional molecular laboratory, pertaining to sample extraction setup and equipment and many aspects of contamination control. In general, they also require only a few minutes of hands-on time per specimen, freeing up lab staff to deal with other duties while the instrument handles the whole process.
All of these attributes make Sample-to-Answer instruments attractive for ‘near POC’ applications, where molecular testing facilities and dedicated staff are not readily available, but samples are, and where 1-2 hour turnaround times on results can be clinically beneficial.
The three key failings of traditional analytics technology as a basis for new generation devices
The need for a new generation of devices is created in the main by the ever-increasing reliance on IVD, its increasing reach into the third world, and the continuing evolution of POC diagnosis. Three factors inherent in the traditional PCR testing process suggest clearly that this can never provide the optimal basis on which to build such devices.
Motors, micropumps and check-valves have moving parts. Their presence in traditional process Sample-to-Answer units designed for POC applications leads to low production yields in fabrication, high failure rates, frequent maintenance need and poor reliability in operation. The machines thus require regular, costly, skilled, and disruptive maintenance.
Complex electromechanical systems and regular maintenance requirements mean that devices based on the traditional PCR testing process have high costs of both fabrication and ongoing operation.
3. Skill Level
The traditional analysis techniques require trained operators in order to perform every step in their procedures without contamination or error.
SAW: The capability to redefine diagnostics
The preparation and presentation of a patient’s sample for PCR testing is the part of the process that creates the need for complex and sensitive electromechanical device components, and so limits the potential for low-cost implementations.
SAW (Surface Acoustic Wave) technology, however, offers an exceptional method for microscopic sample manipulation on surfaces.
Acoustic waves contain a mechanical energy that can be used to manipulate fluids, cells, and samples. This energy is concentrated at the interface between the fluid and the substrate, enabling a range of fluid manipulations on a chip.
SAWs have shown great potential to perform ultrafast ‘microcentrifugation’ on chip, in which particles or cells are concentrated within a fluid. The technique provides a powerful method by which it is possible to handle droplets and particles in a programmable fashion.
For example, droplet movement, merging and centrifugation on the same substrate is possible, with only the need to change the SAW excitation frequency to achieve a high degree of functional integration.
Investigating the use of Surface Acoustic Waves
A Biomedical Engineering research team at the University of Glasgow has carried out extensive work on the acoustic manipulation of fluidics.
While the techniques initially introduced involved either aligning a drop on the edge of a standard interdigitated transducer (IDT), or positioning a gel to partially absorb the SAW reflection, more complex SAW devices were also developed, including those which focused the SAW using circular transducers with a fixed frequency and excitation pathway.
However, these methods necessitated a precise positioning of the droplet and allowed only a single function to be performed by the IDT.
Developing the potential of SAW
Subsequently, the researchers have demonstrated a new concept in SAW microfluidics which combines the use of a disposable glass superstrate with a ‘slanted finger’ IDT.
This technique provides a method by which droplets and particles can be handled in a programmable fashion. The team has demonstrated droplet movement, merging and centrifugation on the same substrate, needing to vary only the SAW excitation frequency to achieve a high degree of functional integration.
While other techniques depend on the input power to control the concentration of particles, the Glasgow team has demonstrated that this can be performed simply by shifting the position of the SAW, and hence its region of interaction with the droplet.
The team has also performed more complex tasks, which were programmed sequentially into a single IDT device. In this demonstration two droplets were moved, merged, mixed, and centrifuged on a disposable superstrate. This evidences the flexibility of the platform for basic fluidic operations needed in lab-on-a-chip technologies.
Demonstrating the viability of SAW-based devices
This represents a new level of capability in microfluidics, showing that microfluidic manipulations, including the centrifugation of blood, can be performed on a disposable phononic chip.
In the team’s demonstration, the SAW excitation frequency was chosen to couple across the substrate–superstrate interface, where droplet manipulation was achieved. The phononic structures interact with the acoustic field, as a ‘filter’ or a ‘waveguide’, providing excellent reflectivity or scattering to the incoming acoustic waves.
The team showed how sample actuation is dependent upon the frequency of the acoustic wave and is able to produce fluid motions on a disposable.
At the next research and development stage, they demonstrated the detection of microbial sequences from blood, using real-time PCR. Acoustically generated vortices in the liquid allowed controlled disruption of blood cells and of blood cells with malarial parasites within them. Changing the excitation frequency resulted in efficient heating of the sample, as a consequence of dissipation of acoustic energy within the drop. Passive cooling and reheating enabled Nucleic Acid based Tests (NATs).
This acoustic sample preparation and PCR demonstrated the ability to detect microbes in a microliter-sized blood sample to the same level of sensitivity achieved in traditional, lab-based PCR tests.
The advantages of SAW based devices
This demonstration not only evidences the ability of SAW actuated technology to detect trace malaria parasitemia in blood on a low cost, low power platform. It also shows the potential of phononics, when coupled with microfluidics, in providing a frequency-dependent toolbox for lab-on-a-chip.
Like other diagnostic techniques, Nucleic Acid based Tests (NATs) usually require well-equipped laboratories with sample preparation apparatus, such as a centrifuge and thermal cyclers to carry out PCR. However these apparatuses can be replaced by microfluidic chips, using various combinations of SAW and phononic technologies.
The method also provides a clear route towards the integration of PCR to detect pathogens in a single handheld system.
The platform also has the potential to be developed towards the detection of other infectious organisms in other samples (such as tuberculosis in sputum), or RNA-based methodologies (viruses using reverse transcription PCR).
The key point is that in any case, the platform enables the whole analysis at modest cost and at low power.
Diagnostic devices utilising a SAW process have a number of significant advantages over those attempting PCR using traditional techniques, be these portable or laboratory devices.
1. No moving components & less maintenance requirement
SAW induced, non-contact, bulk movement of fluid does not involve moving parts. This contrasts with the large, pressure driven pumps required by many fluid manipulation methods, and with the integrated electrodes required for electrokinetic mechanisms, and results in a substantially reduced maintenance exposure.
Phononic devices can be produced at such low cost that it is easy to envision their use with disposable parts in many areas of diagnosis. In this scenario, the SAW would be coupled into a disposable phononic chip, placed within a re-usable instrument, thus providing an extremely low-cost technology.
Further cost benefit, however, lies in the programmability of the chip, as different assay protocols can be realized with the same chip layout and hardware.
Additionally, the low requirement for skilled technicians also indicates an attractive cost benefit, as do the lack of requirement for a clean environment and the low maintenance described above.
The absence of costly consumables also presents a substantial saving advantage over the current generation of cartridge-based POC and laboratory devices.
3. POC with no need for professional operators
Despite being an automated system, as above the absence of moving parts in a SAW device significantly decreases the need for maintenance. Additionally, with an automated system capable of running the entire process, starting from a raw sample such as a swab, a relatively low qualified individual can operate an automated SAW engine making it well suited to POC use.
The SAW technique has particular application to the noncontact manipulation of liquid droplets, for example in biological, biochemical, medical, veterinary, and chemical assays, analysis, diagnosis, and synthesis and production of reagents and chemicals.
A waveguide and filter sample preparation unit has been designed for lysing cells and pathogens and for fluid/reagent manipulation, both exploiting the efficient use of acoustic energy.
Compared to the other sample manipulation technologies, such as magnetic tweezing, dielectrophoresis and optical tweezing, ultrasonic manipulation has shown potential in a variety of applications, with its advantages of versatile, inexpensive, and easy integration into microfluidic systems, with low power consumption.
Apart from time saving and cost reduction, the system combines highest sensitivity and reliability towards a fully automated lab-on-a-chip in the expanding field of point-of-care diagnosis and individualised therapies.
Through the University of Glasgow’s commercialisation unit, the Glasgow team invites discussion over licensing, collaborations, further R&D, investment, and other, similar opportunities.
The team is especially interested in discussions with diagnostic and medical device manufacturers, as well as laboratories and healthcare services providers to whom the potential for low cost, lab-on-a-chip POC IVD devices might be of interest.
In short, the team’s door is open to anyone with an interest in employing its IP to transform the diagnostics landscape, and in benefitting from the significant potential for commercial benefit to be obtained from so doing.
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For more information on this technology, contact Darian Brookes
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About the inventors
The SAW research is led by Professor Jonathan Cooper and Dr Julien Reboud within the Biomedical Engineering Research Division at the University of Glasgow. Professor Jonathan Cooper holds The Wolfson Chair in Biomedical Engineering. Dr Reboud is Senior Lecturer in the James Watt School of Engineering.
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