Lieven Gentaur
News
What are Digital and Absolute Assays?
Digital assays refer to formats in which individual target molecules (or events) are counted as discrete units (often “0 or 1” per compartment or detection event) rather than measuring a continuous averaged signal. This enables more precise quantification of low-abundance species. For example, digital immunoassays for proteins can detect attomolar concentrations by partitioning and counting positive/negative events.
Absolute assays mean that the read-out corresponds directly to the number of target molecules (or well-defined units) present in the original sample, without reliance on a calibration curve or relative standard to infer concentration. This is sharper than “relative quantification” and allows proper comparisons between samples and across labs.
Why Flow Cytometry (FC) Matters in This Context
Traditionally, flow cytometry is used to count cells or particles (e.g., immune-cells, bacteria, beads) by fluorescence or scatter, yielding population percentages or relative abundances. But recent innovations push it into the molecular domain:
- Flow cytometers are inherently high-throughput (thousands of events per second) and capable of multiplexed fluorescence detection (multiple channels, colours).
- By adapting optics, microfluidics and labeling strategies, flow cytometers (or custom “digital flow cytometers”) can detect individual molecules or small assemblies in flow enabling absolute counting in a suspension format rather than on a surface or in droplets alone. For example, a recently described “digital flow cytometer” (dFC) demonstrated single-molecule detection efficiency >98% and a dynamic range of ~4 orders of magnitude.
- In your field of microbial biomarker detection, flow cytometry offers the ability to handle complex, heterogeneous samples (e.g., hospital surface swabs, bacterial/viral particles, extracellular vesicles) and offers multiplexing plus statistical power of high event counts.
Thus, integrating digital/absolute assay concepts with flow cytometry offers a powerful route to detect very low-abundance biomarkers with high specificity, throughput and quantitative accuracy.
Key Technological Developments & Practical Considerations
Sensitivity & detection limits
Digital assay strategies can push limits of detection (LOD) to attomolar or femtomolar levels for molecules. For example, review work on digital protein detection shows immunoassay sensitivities down to ~10 proteins in 200 µL samples. Flow-cytometry adaptations (e.g., dense fluorescent labeling, high numerical aperture optics) have achieved single-molecule quantification in flow with LODs ~47 fM for microRNAs.
Absolute quantification & dynamic range
In the dFC study, the system achieved accurate concentration measurements with R² values ~0.999 across serial dilutions. That level of precision and dynamic range is critical when biomarkers may span several orders of magnitude (e.g., from extremely rare to moderate abundance) and when you need to compare across samples or time.
Multiplexing
One of the advantages of flow cytometry is multi-parameter detection (multiple fluorophores, detectors). Digital flow systems are increasingly implementing multi‐lasers and channels to detect multiple targets simultaneously. For example, the dFC system had 4 spatially separated lasers and 12 detection channels. Multiplex detection of biomarkers (e.g., several resistance genes, virulence proteins, microbial toxins) is highly relevant in your domain.
Sample matrix and target preparation
Low‐abundance biomarker detection in complex matrices (hospital surface swabs, environmental fluids, bacterial lysates) requires careful attention to sample preparation: efficient capture, minimal loss, reduction of background/non‐specific signal, good labeling strategies. Digital systems are particularly sensitive to losses or background because when your target count is very low, any inefficiency or noise becomes a large fraction of signal.
Workflow, throughput & cost
Digital/absolute assays (especially early or custom systems) may have added complexity (microfluidics, specialized optics, careful calibration). Flow cytometry offers familiar workflows (suspension samples, lasers, detectors) and the potential for higher throughput which is advantageous in research/monitoring of many samples (e.g., hospital surfaces, microbial surveillance). But such systems still require optimization, controls, and method validation.
Statistical considerations
When counts are low, Poisson statistics dominate the sampling error (variance ~mean) matters. For absolute quantification, you must ensure enough input volume, good capture efficiency, and account for losses so that counts reflect original sample. Many digital assay reviews caution on this.
Application to Low-Abundance Molecular Biomarkers in Microbiology & Hospital Environment
Given your experience with hospital environments, antimicrobial resistance, surface sampling and microbial detection, here are how digital/absolute flow cytometric assays could impact your work:
Detection of rare resistance gene transcripts: Suppose you sample hospital surfaces for bacteria harboring a particular gene (e.g., carbapenemase gene) or its transcript. Traditional qPCR may give Ct values, but absolute counting of molecules (via digital flow assay) would allow you to say “X molecules per swab area” and compare across time, surface types or disinfectant protocols.
Quantification of low-abundance bacterial proteins related to disinfectant stress: For bacteria on surfaces, you might target stress‐response proteins in low copy number. A flow-cytometric digital assay could label single bacterial lysates/bead‐coupled proteins and count events, giving true molecule numbers rather than relative fluorescence.
Multiplex monitoring of multiple microbial biomarkers: For example, tracking efflux pump proteins + biofilm markers + resistance enzyme transcripts from the same sample. A multiplex digital flow cytometric assay could count each target independently.
Time-series or longitudinal monitoring: In retrospective or surveillance studies of hospital surfaces, you can quantify how the abundance of biomarkers (gene copies, proteins) changes over time, correlates with disinfection protocols or outbreak events absolute counts provide better comparability than relative units.