Sheath-enhanced concentration and on-chip detection of bacteria from an extremely low-concentration level

Abstract

Microfluidic-based sheath flow focusing methods have been widely used for efficiently isolating, concentrating, and detecting pathogenic bacteria for various biomedical applications due to their enhanced sensitivity and exceptional integration. However, such a microfluidic device usually needs complicated device fabrication and sample dilution, hampering the efficient and sensitive identification of target bacteria. In this study, we develop and fabricate a sheath-assisted and pneumatic-induced nano-sieve device for achieving the improved on-chip concentration and sensitive detection of Staphylococcus aureus (MRSA). The optimized nanochannel design with diverging configuration is beneficial to the regulation of the hydrodynamic flow while the sheath flow is focusing the sample to the confined region as expected. Per the experimental finding, a high flow ratio (sheath flow/sample flow) presents enhanced target concentration by comparing with a low flow ratio. With this setup, MRSA bacteria with an extremely low concentration of ∼100 CFU mL^-1 are successfully and sensitively detected under a fluorescence microscope, less than 30 min, demonstrating a reliable sheath-enhanced concentration and on-chip detection for target bacteria. Additionally, the theoretical model introduced here further rationalizes the working principle of our nano-sieve device, potentially guiding the optimization of next generation devices for highly sensitive and accurate on-chip bacteria detection at a much lower concentration level below 100 CFU mL^-1.

Fig. 1

(a-i) The schematic of the sheath-assisted and pneumatic-induced nano-sieve device presenting an overview of channel configuration. (a-ii) The local region of MRSA–bead interface. (a-iii) MRSA physically concentrated by stacked beads with diameters of 10 μm and 1 μm. (b) Fabrication procedure of the device by plasma bonding the PDMS pneumatic chamber, PDMS thin film, and channel-patterned glass substrate. (c) The optical picture showing the experimental device with low and high magnifications.

Fig. 2

(a) The bright-field pictures showing the profile of the focusing stream caused by the sheath flow (a-i) and the formation of bead stacking in the channel (a-ii). (b) The visualization of the focused mainstream displaying the relationship between the “working width” and flow conditions. The inset picture shows the sketch of the designed nano-sieve channel. (c) The linearity of the measured width as a function of flow ratio for both theoretical calculation and experiments.

Fig. 3

The concentration effect under various sheath flow/main flow conditions. (a) The fluorescence images visualize the measured “working width” related to the various flow conditions of low ratio (a-i), middle ratio (a-ii), and high ratio (a-iii). (b) The result comparison between the theoretical model and experiments. (c) The quantitative data present the concentration effect under different flow conditions. (d) The initial sample solution of fluorescent nanoparticles (1.71 × 10⁵ beads per mL) detected on a glass slide. (e) The concentrated nanoparticles were observed at (e-i) 4-fold magnification and (e-ii) 20-fold magnification, respectively.

Fig. 4

The fluorescence signal monitoring for accumulated fluorescent nanoparticles (green) at the specific region. (a) Bead stacking within the nano-sieve channel under the high flow ratio condition. (b) The fluorescence images presenting the concentrated target nanoparticles for the processing times of (b-i) 5 min, (b-ii) 10 min, (b-iii) 15 min, (b-iv) 20 min, (b-v) 25 min, and (b-vi) 30 min. (c) The measured integrated density as a function of elapsed time in a total time of 30 min.

Fig. 5

The image panel exhibits the comparison of on-chip and off-chip results under the fluorescence microscope. (a) MRSA bacteria sample solutions with three different MRSA concentrations of (a-i) 100 CFU mL⁻¹, (a-ii) 10³ CFU mL⁻¹, and (a-iii) 10⁴ CFU mL⁻¹, were placed on glass slide. (b) MRSA bacteria sample solutions with three different MRSA concentrations of 100 CFU mL⁻¹ (b-i), 10³ CFU mL⁻¹ (b-ii), and 10⁴ CFU mL⁻¹ (b-iii), were captured within the nano-sieve channel. The stained MRSA bacteria present blue fluorescence. Blue arrows indicate the detected targets. The inset shows the bacteria identification with higher magnification.