28 June 2024

Authors: Dr Andrew Breeson and Prof Atul N. Parikh

Traditional (analogue) assays have been crucial in advancing scientific understanding and clinical diagnostics, but they come with limitations that can affect the accuracy, efficiency, and scalability of biological testing. Today, digital assays are transforming our approach to diagnosing diseases and reshaping healthcare by allowing researchers to detect and analyse single molecules in messy, crowded, and heterogeneous biological environments. This blog discusses some of the main drawbacks of traditional biological assays and gives real-world examples of how digital assays can overcome them.

Most traditional assays are not sensitive enough to detect very low concentrations of biomolecules. This limitation can be critical when diagnosing clinical conditions or diseases early on using blood-based biomarkers or detecting low-abundance environmental toxins. In both cases, target molecules can be present in samples at vanishingly low (femto-, atto-, or zeptomolar concentrations) corresponding to as few as 1-100 copies per microlitre of the sample.   

Digital bioassays break this barrier by pushing the limit of detection to single-molecule levels to offer significantly higher sensitivity. These assays achieve this remarkable feat simply by (1) dividing the sample into fractions, isolating individual molecules or particles in microwells or droplets, and (2) digitally amplifying otherwise weak signals corresponding to single molecules.

Consider, for example, the case of early diagnosis of a disease or a clinical condition such as cancer by “liquid biopsy.” Some of the earliest biomarkers are circulating tumour DNA fragments and circulating microRNA, present in various biofluids (blood, urine, cerebrospinal fluid, and pleural fluid) at sub-attomolar concentrations. Digital bioassays can isolate and detect these trace concentrations of tumour DNA in a patient’s blood (or other) sample. This heightened sensitivity allows for cancer detection at a much earlier stage when treatment options are more effective, and the chances of a favourable prognosis are significantly higher.

Analogue assays often suffer from variability in measurement and limited quantitative precision. Factors such as operator technique, environmental conditions, and the quality of reagents can influence the results, leading to less reproducible results.

Digital assays use advanced imaging and computing technologies to count every single discrete event (like individual molecule interactions). Because these assays count each event, they need (in principle) no calibration or benchmarking and can serve as absolute assays. Therefore, they produce highly precise and reproducible quantitative data (however, there are practical limitations when the assay fails to count all of the targets in the sample volume).

This quantitative precision can be crucial. For instance, during a viral outbreak, public health officials rely on precise diagnostic data to track the spread of the virus and implement control measures. Variability in analogue assay results can lead to incorrect estimates of infection rates, hampering efforts to contain the outbreak. Digital assays, with their high precision and reproducibility, provide reliable data that public health officials can trust. The precise quantification offered by digital assays allowed for early detection of infection surges, enabling timely public health responses and interventions.

Traditional assays typically need relatively large sample volumes, which can be a drawback in situations where only small amounts of biological material are available.

By leveraging micro- or nano-scale technologies, digital bioassays require much smaller sample volumes. This conserves precious or rare samples and reduces reagent costs.

For example, in paediatric oncology, obtaining large blood samples from young patients can be challenging and distressing. Digital bioassays, which can perform highly sensitive analyses on minute blood volumes, allow for comprehensive diagnostic tests and monitoring with minimal sample collection. This not only eases the burden on young patients but also ensures that critical information about their health is gathered efficiently and accurately. Additionally, in research involving rare or endangered species, where biological samples are limited, digital assays enable scientists to conduct extensive studies and obtain reliable data without depleting the scarce resources available.

Many traditional bioassays are labour-intensive and time-consuming, requiring significant manual labour for setup, monitoring, and analysis. This can lead to delays in obtaining results and increased costs.

Digital bioassays are amenable to automation and integration for sample handling, processing, and analysis. This automation speeds up the assay process, minimises labour-intensive tasks, and eliminates handling errors, together leading to faster turnaround times for critical diagnostics.

During a disease outbreak, rapid and accurate diagnostics are essential for effective response and containment. Automated digital PCR systems can quickly process and analyse large numbers of samples with minimal human intervention, providing timely results that are crucial for identifying and isolating infected individuals. This efficiency not only reduces the workload for laboratory personnel but also ensures that healthcare providers receive critical information faster, improving patient outcomes and public health responses​​.

Scaling up traditional assays for high-throughput screening can be challenging and costly, as it often requires extensive re-optimisation and can increase the risk of errors.

Digital bioassays are designed to be easily scalable, allowing for the simultaneous processing of thousands to millions of micro-reactions. This scalability is advantageous for high-throughput screening applications in drug development and genetic testing.

In the pharmaceutical industry, drug discovery processes often involve screening vast libraries of compounds to identify potential therapeutic candidates. Traditional assays may struggle to handle the volume efficiently and accurately. Digital bioassays, however, can simultaneously evaluate the effects of millions of compounds on target proteins or cells by partitioning the reactions into microdroplets. This approach not only speeds up the drug discovery process but also increases accuracy and reduces costs, ultimately accelerating the development of new medications​​​​.

In conclusion, digital assays represent a significant advancement in the field of molecular diagnostics and analytics. At IDMxS, we are at the forefront of this revolution, harnessing the power of digital molecular analytics to push the boundaries of science and improve global health outcomes. To learn more about our work, click here.

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