Advances in precision medicine and diagnostics over the past decade have the potential to improve patient outcomes by decreasing the time needed to identify a disease or pathogen and adjust patient treatment, whether they are to address rising cases of antibiotic resistance, viral epidemics or facilitate more tailored cancer therapy. The ever-accelerating development in this area can be attributed to rapid advances in microfluidic devices, nucleic acid amplification, synthetic biology, battery technology, automation, sensors, bioinformatics and data analysis.
Regardless of the operating principle, developing a new diagnostic can present a variety of technical challenges during the transition from a lab workflow to a finished product. These depend on the intended use and use environment of the device as well as the underlying technology and its amenability to a scalable manufacture and low cost.
One aspect is universally applicable to the development of a wide variety of systems. Sample collection and isolation represent crucial stages in diagnostic system workflows regardless of the analytes being detected or the diagnostic principles of the instrument itself (1). Due to the diverse nature of samples, there’s no one-size-fits-all approach, and many different methods have been developed for use in the wide variety of settings for which they are needed (1, 2, 3). Extracting isolates from multi-phase, complex samples poses inherent challenges as they resist easy separation. This poses limitations on what can be analysed particularly with smaller point-of-care (POC) systems as these are limited in terms of power, system complexity, footprint and cost. Even automated laboratory systems can struggle with the complexity of isolation protocols, requiring manual preparation at the cost of resource. Sample preparation is estimated to account for approximately 66–80% of sample analysis time, and is frequently the source of error and variability in a protocol because of its knock-on effect on sample purity and detectability (1).
The development of effective and scalable isolation technologies is therefore critical for advancing an effective and versatile diagnostic technology. Addressing these challenges in development greatly enhances the capabilities of the system and future-proofs future assays and diagnostic panels. In this article, we delve into common challenges encountered during sample preparation and the implications on system and consumable design. By understanding these challenges, we can pave the way for more robust and efficient diagnostics which have the potential to significantly aid capability at the point-of-care.
The challenge
The physical properties of a sample to a diagnostic system can vary from mucus, urine, blood, stool or even solids such as biopsy samples (3), as will the target analyte depending on the application. Even in the context of the most common biochemical or molecular techniques the strategy for pre-treatment (separation and concentration) of analytes from these samples can be diverse, as they have very different composition and analyte concentration. Further preparation through chemical modification is also required before detection and analysis can take place, and contaminants from upstream processes can interfere with later stage detection, reducing sensitivity and specificity.
State of the industry
Immunoassays or nucleic acid amplification tests are some of the most widely used biochemical and molecular diagnostic methods, particularly ELISA and PCR (2). Most preparation methods for these systems rely on the use of organic solvents immiscible in water to extract analytes from a sample e.g. liquid-liquid extraction (LLE)(1). The use of solvents poses a number of challenges due to their toxicity and in the context of a polymer consumable, reactivity and volatility which can limit shelf life of the consumable element. In response to these challenges, various innovative techniques have been developed including spray and electromagnetic systems. The area of solid-phase extraction (SPE) builds on typical workflows but minimises solvent usage while streamlining the underlying processes. Unlike LLE, SPE does not require mixing of two liquid phases, rather the liquid sample is passed through an absorber (usually silica or polymer based) to which the analytes have a greater affinity. Subsequently, the analytes are extracted by elution with an appropriate solvent. The absorber can function in a variety of ways including phase separation, selective sorption, ion exchange and complex mixed-mode media including molecularly imprinted polymers (MIPs) (5,6). MIPs are synthetic materials with manufactured binding sites, mimicking biological counterparts such as antibodies known for their sensitive and selective affinity. MIPs are extremely powerful in biomarker isolation from biofluids. The use of SPEs such as these greatly reduces the volume of solvent required and lends itself to automation and miniaturisation (4, 5). Indeed, recent developments have integrated SPE directly into 3D-printed microfluidic chips aimed at analysing biomarkers related to preterm birth demonstrating how such technology can aid rapid bioanalysis of medically relevant analytes (7). Pathogen identification from blood is one example where the sample can affect successful isolation as nucleic acid amplification by PCR in the presence of blood components has been problematic. Because even low concentrations of bacteria in the bloodstream can lead to sepsis, sample volumes need to be large, and this has implications on the separation technology used as some are flowrate limiting or lack the necessary selectivity.
Techniques with the most promise include screening, sedimentation, and affinity capture through the use of SPEs and magnetic beads, as they allow faster processing of larger sample volumes (8). Complex samples such as stool or biopsy present a different challenge due to their variability and heterogenous composition which can create a problem for creating preferential affinity for an absorber. Often a homogenization and separation step is required before isolation of analytes can take place, strong affinity between phases also limits the recovery efficiency. This area of innovation is quickly advancing with various techniques including heating, ultrasonic and mechanical methods have been proposed to overcome present challenges (9). A recent report demonstrated a novel on-chip method for liquify stool samples using acoustic streaming (On-chip stool liquefaction via acoustofluidics). The device employs an acoustic transducer to generate strong micro-vortexes, homogenizing stool samples at flows rates up to 30 µL/min while filtering out larger debris using on-chip micropillars. Notably immobilised enzymatic methods have the greatest potential for not only providing a low-power passive solution but also more sustainable green methods (1) (10). This has been exemplified in paper-based microfluidic analytical devices (11) which show promise as a low cost, disposable, rapid and easy to use diagnostic tool employing immobilized enzymes to detect pathological biomarkers (12).
Implications of integrated preparation on system architecture
Even with advances in materials and reagents, a challenge remains in successfully implementing sample preparation and isolation workflows within integrated or automated systems. Microfluidic consumables can offer the ability to automate complex workflows and eliminate operator error, using smaller, precisely dosed volumes. However, if workflow complexity if not optimized in the early stages of development it can lead to an overcomplicated architecture and thereby instrument interface. Each level of complexity presents additional risk and can result to excessive dependence on geometric tolerances which are not easily manufacturable. Geometric tolerances on multi-component stacks can negatively affect sealing, fluid path integrity and stability, as well as sample portioning and chamber filling rates.
The approach to integration a sample preparation system should be holistic and be driven as much by the target application as it is by the resources available within the target product profile.
Using system components to perform multiple functions can significantly reduce overall complexity at the cost of component complexity. For example, using the fluid transfer system for spatial chromatographic separation, exploiting innovative microfluidic designs for precise flow control and enhanced analytical capabilities in chemical and biological applications (13). Another example is the use of a metalized filter for bacterial separation which can also function as a heating element for subsequent lysis and amplification. The solution as always is to keep it simple, relying on the development of another complex technology to facilitate an already novel and complex technology only compounds risk, where existing systems and approaches maybe a more robust and equally customizable solution.
Conclusion
Sample collection and isolation are critical parts of diagnostic workflows, which require an adaptable approach due to the diversity of sample types. Challenges such as isolating analytes from multi-phase samples and the need for chemical modification underscore the critical role of effective isolation technologies. While miniaturisation and the use of microfluidics can automate and de-risk some of these workflows, successful designs necessitate a holistic approach, which considers both the purpose or application and the available resources in terms of space, cost power and user expertise. Simplifying complexity at the workflow level through multifunctional system components can mitigate risks associated with geometric tolerances and overall system architecture. Ultimately, a balance of system level and component level simplicity is key, leveraging existing technologies to enhance a diagnostic systems efficiency and robustness. Addressing these challenges and embracing innovative yet pragmatic solutions paves the way for more resilient and efficient tools, empowering healthcare professionals, and improving patient care in a wide variety of settings.
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