A Microfluidic Approach for High-Throughput Raman Spectroscopy of Whole Blood

Raman spectroscopy is an optical technique used to probe the molecular composition of a sample. It relies on the inelastic scattering of light: when a laser interacts with molecules, a small fraction of the scattered light undergoes a shift in energy that is characteristic of the vibrational modes of the molecules. This spectral fingerprint enables the identification and analysis of chemical species without the need for dyes or markers. [2,3]

Because of its specificity and non-destructive nature, Raman spectroscopy is widely used across multiple fields, including chemistry, materials science, and biomedical diagnostics. In particular, it has gained strong interest for biological applications such as cell analysis, pathogen detection, and blood diagnostics, where label-free and real-time measurements are highly valuable (Figure 1). [2,3]

However, despite its potential, Raman spectroscopy faces several key limitations. The Raman signal is inherently weak, leading to long acquisition times and limiting throughput. In complex and non-transparent samples, signal attenuation and background noise further degrade performance. [2,3]

The microfluidic chip was fabricated in fused silica. It employs 3D hydrodynamic focusing to confine and center the sample stream within a surrounding buffer flow, enabling precise control of the stream size (down to 15 µm) and optimal alignment with the Raman excitation volume. A locally thinned observation region enhanced signal collection while reducing background noise. This design also supported parallel flow multiplexing, allowing high-throughput and simultaneous analysis of multiple samples (Figure 2).

To ensure stable and precise flow control,the microfluidic set-up relied on a pressure-driven system composed of a Fluigent MFCS pressure controller which regulates the sample and buffer reservoirs, along with two Fluigent Flow Unit sensors for real-time monitoring of flow rates upstream of the chip. By adjusting the pressure ratio between the sample and sheath flows, the sample stream was hydrodynamically focused at the center of the microchannel.

Raman measurements were performed using a 532 nm continuous-wave laser focused into the microchannel through a high-NA objective, which also collected the backscattered signal. The Raman signal was separated from the excitation light using dichroic and notch filters, then analyzed via an imaging spectrometer coupled to a cooled CCD camera.

Proof of Concept: Microfluidic Raman Analysis of Whole Blood

The study showed improved performance enabled by the microfluidic approach compared to conventional capillary-based measurements. As displayed in Figure 3, precise hydrodynamic focusing allowed the sample stream to be confined down to 15 µm and matched to the excitation volume, enabling full-volume interrogation within a single measurement. This resulted in a linear relationship between Raman signal intensity and analyte concentration (Figure 4), confirming the system’s quantitative capability down to micromolar ranges.

When applied to whole blood, the impact of microfluidics becomes more observed. In Figure 5 and Figure 6, the microfluidic chip enabled signal collection from nearly 100% of the flowing sample, whereas capillary-based measurements probe only a small fraction (8%), with significant background contribution from the walls.

This improved sampling efficiency, combined with uniform flow conditions at the channel center, leads to more representative and reproducible measurements, especially for heterogeneous or corpuscular fluids. Therefore, the platform enabled acquisition of Raman spectra to be more representative of the full cell population, which is particularly critical for detecting rare pathological cells.

Importantly, this microfluidic configuration enhances throughput while reducing photodamage. The higher flow velocity increased the number of cells analyzed per unit time, while the reduced interaction time allowed the use of higher excitation powers without inducing degradation. This resulted in stronger signals and shorter acquisition times, demonstrating the clear benefit of microfluidics for high-throughput Raman analysis.