75) in backscattering mode. An Ar+ laser (Coherent, INOVA 70C Series Ion Laser, Santa Clara, CA, USA) provided the excitation source v = 514.5 nm. Measurements were conducted with a 200 ��m slit and 100 ��m confocal hole. For SERS measurements, laser power was reduced from 100 mW to 10 mW using a neutral filter with an optical density of 1. The full spectra were acquired in three spectral windows for total acquisition time of one minute. Optical micrographs were recorded using an Axioskop microscope (Zeiss, Jena, Germany) with an external light source (Illuminator, Cole-Parmer Canada, Montreal, QC, Canada). A home-built polycarbonate holder was used to accommodate the fluidic connections and achieve the proper orientation for Raman and optical inspection.Raman and UV-Vis spectra were treated and analysed using Grams/AI 8.

0 for baseline correction, peak deconvolution and intensity measurements. Optical density data were extracted from micrographs using the open source software ImageJ V1.47.For descriptions of processes related to bacterial culture, system sterilization, inoculation and biofilm culturing, readers are referred to the section on biological materials preparation in the Supplementary Materials of this paper.2.1. Fabrication of a Two-Level Bioreactor for Flow Confinement against the SERS SurfaceThe present microbioreactor was a two-level system (Figure 1A) fabricated in PDMS. The channel structures were fabricated by casting uncrosslinked PDMS against a silicon mould with patterned photoresist features.

These features Brefeldin_A had the inverse dimensions of the required channels, but resulted in the required channel dimensions in the PDMS following casting. Levels 1 and 2 consisted of channels with dimensions of width w = 2 mm, height h = 305 ��m and length l1 = 32 mm and l2 = 9 mm, respectively (Figure 1B). The two levels were aligned and bonded such that the channels therein were collinear and there was overlap between them. A cylindrical junction was formed between the overlapping segments using a punch (diameter = 500 ��m). The punch angle was 45 degrees, such that the liquid entering the channel in Level 1 had some component of its velocity in the x-direction in order to: (i) keep the biofilm precursor stream close to the bottom of the Level 1 channel; (ii) reduce shear forces between the two streams and (iii) maintain smooth laminar flow. Level 1 channel was sealed by a glass cover slip with thickness of 170 ��m, which matched the working distance of the Raman spectrometer system. Confining liquid (pure water) and biofilm precursor liquids (bacterial inoculants and citrate solutions) were introduced into Level 1 and Level 2 channels via Inlet 1 and Inlet 2, with a flow rate Q1 and Q2, respectively.