The Challenges of Developing Ultra-Sensitive Diagnostic Assays

An expert describes the technical hurdles behind designing the next generation of in vitro diagnostics systems. 

Nick Collier, PhD

Ultra-sensitive diagnostic assays are used to diagnose and detect the early onset of disease. A new report by Technavio projects the global high-density multiplexed diagnostic assays market reaching $1.67 billion by 2021. According to the report, just some of the factors propelling the market to grow at a compound annual growth rate of 15% include:

  • A rise in the incidence and prevalence of autoimmune, oncology and cardiac disease;
  • higher adoption of point-of-care testing systems;
  • and a greater focus on personalized medicine.

Sagentia is at the forefront of research and development in clinical diagnostics, having completed more than 500 IVD projects for our clients ranging from low-cost home-use devices to single molecule sequencing platforms. These are some of the technical challenges which should be considered when designing the next generation of in vitro diagnostics systems. 

The Trend to Small Sample Volumes

As testing moves closer to point-of-care and outside of a clinical setting, it becomes desirable to reduce the volume of a blood sample required to run an assay. Smaller sample volumes make blood collection easier from the patient and potentially allows for finger prick samples. While there is some debate as to whether capillary blood is equivalent to a venous draw, for several assays the advantages of the former are significant and clinical trials are expected to show it can be done. However, using small samples demands sensitive assays and new techniques for manipulating such small volumes of fluid.

Technical Challenges of Testing Small Samples

Maintaining a good coefficient of variation at small volumes requires very precise fluid manipulation of both sample and reagents. This becomes very apparent at 10uL, where a 1uL error in sample volume gives a 10% variation immediately if there is no calibration. Sample volumes of 10uL or less are strongly influenced by surface tension and surface properties of the cartridges and plasticware to the extent that samples do not always end up where you want them to be. Accurate pipetting is possible at small volumes, but care and skill is required and evaporation must also be considered if there are lengthy incubation steps.

Microfluidic Solutions to Small Sample Testing

The trend to small sample volumes and the need for easy-to-use PoC tests is driving the development of microfluidic solutions that move away from the classical method of pipetting and robotics. Pneumatically, centrifugally and capillary-driven flows in microchannels are one such example which get around some of the challenges. Digital microfluidics using electrowetting and acoustic manipulation of droplets can manipulate small volumes and perform complex multistep sample properties workflows. These emerging new approaches overcome some of the technical challenges of small sample testing. Within these devices, we also see the use of acoustic streaming, magnetics, electrophoresis, and dielectrophoresis to manipulate cells and particles.

Difficulties with Microparticles and Small Volumes

Microparticles are a powerful tool for assays, providing high surface areas and short diffusion lengths for the selective capture of molecules and cells of interest. To be used effectively, it is necessary to achieve good mixing, capture, and resuspension of the particles. The use of these particles in 96-well and 384-well formats, for example, is well developed and often performed using a three-axis robot emulating a manual technique. However, the approach works less well for single PoC tests and is challenging to use for small sample volumes.

A range of factors can impact the accuracy and sensitivity of the assay. Beads are captured from suspension by a magnetic field. It takes time for the beads to travel to the magnet so, with shorter collection times, the less efficient the process will become and beads will be lost. While the percentage of beads lost might be very small, it can become significant if 10–20 cycles are required, as is the case in immunoassays for example.

After collecting the beads and removing supernatant, the beads usually must be resuspended. Poor resuspension of the beads may occur. If the beads do not resuspend well after capture this can result in poor capture, poor washing, or some beads not being exposed to the full concentration, which could subsequently skew the results. Most assays require the beads to be mixed well with the reagents and sample. This is normally achieved using a process of vortexing. Though convenient on the lab bench, vortexing becomes more challenging as sample volumes are reduced.

Microfluidic Solutions

There are several alternatives to traditional resuspension and pelleting. In microfluidic solutions, it becomes possible to move the fluid past the dispersed beads rather than moving the beads through the fluid. This has the advantage of providing different options for designing the consumable. In small sample volumes, ultrasonic streaming is an effective method of moving the beads. Instead of bringing fluids in and out of a vessel containing the beads, it is possible to move the beads between different volumes of fluid using magnetic fields, as with superparamagnetic bead manipulation.

CLIA-Waiver Requirements

As tests move from centralized laboratories to regional labs and then into clinics, the burden of sample preparation increases along with the risk it will be performed incorrectly. If the test is intended to be used by untrained or unskilled users outside of a controlled environment it will need a CLIA waiver. If so, it must be simple and have a low risk of an incorrect result.

High-Volume Manufacturing Cartridge Design

As point-of-care testing becomes the norm, other design considerations are needed. The economies of scale for POC testing systems must work, with commercial success often dependent on achieving a sufficiently low cost per test.  This puts pressure on the design and cost of producing the consumable element. That makes it crucial to consider where to separate the core instrumentation (or platform) and the disposable consumable.

Early cartridge prototypes are essential to optimize the assay chemistry, the cartridge, and the instrument design. Often this requires the use of the final materials like polymers and surface coatings, making it challenging to use rapid prototyping techniques such as 3-D printing. It might be more cost- and time-effective to produce low-volume mold tools.

Other System Design Considerations

The range of tests that can be carried out by a IVD system will determine the size and type of the market it can address.  Many healthcare settings will not want an instrument taking up valuable space for just a single test. So, the system’s ability to scale and expand may also be key to it remaining future-proof.

Many tests require onboard reagents to simplify performance of the test without the need to maintain bulk reagents. Where there are onboard reagents, these need to meet stringent shelf-life requirements and keep fluid and dried reagents completely separated until use. Good design will also need to include considerations around the relative water and oxygen permeability properties of the different materials in use.

Finally, if the user introduces the sample into the cartridge, there must be means to control the volume of sample delivered and avoid cross-contamination from sample to sample and from cartridge to operator. Variation in sample volume often translates directly into measurement variation. This problem can be managed in many ways including, for example, designing the cartridge to transfer a defined volume from an input reservoir. Alternatively, it may be possible for the assay to include a reference measurement, against which the instrument compares.

The next generation of in vitro diagnostics systems holds great promise, but also presents great challenges to system developers.

Nick Collier, PhD is chief technology officer at Sagentia. He can be reached at info@sagentia.com.

[Image courtesy of STUART MILES/FREEDIGITALPHOTOS.NET]

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