Nanogap traps for passive bacteria concentration and single-point confocal Raman spectroscopy.

A microfluidic device enabling the isolation and concentration of bacteria for analysis by confocal Raman spectroscopy is presented. The glass-on-silicon device employs a tapered chamber surrounded by a 500 nm gap that serves to concentrate cells at the chamber apex during sample perfusion. The sub-micrometer gap retains bacteria by size exclusion while allowing smaller contaminants to pass unimpeded. Concentrating bacteria within the fixed volume enables the use of single-point confocal Raman detection for the rapid acquisition of spectral signatures for bacteria identification. The technology is evaluated for the analysis of E. cloacae, K. pneumoniae, and C. diphtheriae, with automated peak extraction yielding distinct spectral fingerprints for each pathogen at a concentration of 103 CFU/ml that compare favorably with spectra obtained from significantly higher concentration reference samples evaluated by conventional confocal Raman analysis. The nanogap technology offers a simple, robust, and passive approach to concentrating bacteria from dilute samples into well-defined optical detection volumes, enabling rapid and sensitive confocal Raman detection for label-free identification of focused cells.

In situ photografting during direct laser writing in thermoplastic microchannels.

A method for in situ photografting during direct laser writing by two-photon polymerization is presented. The technique serves as a powerful approach to the formation of covalent bonds between 3D photoresist structures and thermoplastic surfaces. By leveraging the same laser for both pattern generation and localized surface reactions, crosslinking between the bulk photoresist and thermoplastic surface is achieved during polymerization. When applied to in-channel direct laser writing for microfluidic device fabrication, the process yields exceptionally strong adhesion and robust bond interfaces that can withstand pressure gradients as high as 7 MPa through proper channel design, photoinitiator selection, and processing conditions.

Plasma Isolation in a Syringe by Conformal Integration of Inertial Microfluidics.

A thermoplastic microfluidic substrate is conformally integrated onto the cylindrical barrel of a conventional venipuncture syringe, forming a spiral inertial separation element supporting the isolation of plasma from diluted whole blood. The cylindrical shape of the syringe itself serves to define the flow path required for inertial separation by transforming a linear microchannel to a spiral topology. The hybrid system enables inertial plasma separation by Dean flow focusing within the same syringe used for a patient blood draw, with the seamlessly interconnected microfluidic element operated by automated or manual actuation of the syringe plunger. Plasma isolation is achieved without the need for external instrumentation. Device design and fabrication challenges are discussed, and effective plasma isolation within the system is demonstrated, with a peak separation efficiency above 97% using 25 × diluted blood.

Isolation of intact bacteria from blood by selective cell lysis in a microfluidic porous silica monolith.

Rapid and efficient isolation of bacteria from complex biological matrices is necessary for effective pathogen identification in emerging single-cell diagnostics. Here, we demonstrate the isolation of intact and viable bacteria from whole blood through the selective lysis of blood cells during flow through a porous silica monolith. Efficient mechanical hemolysis is achieved while providing passage of intact and viable bacteria through the monoliths, allowing size-based isolation of bacteria to be performed following selective lysis. A process for synthesizing large quantities of discrete capillary-bound monolith elements and millimeter-scale monolith bricks is described, together with the seamless integration of individual monoliths into microfluidic chips. The impact of monolith morphology, geometry, and flow conditions on cell lysis is explored, and flow regimes are identified wherein robust selective blood cell lysis and intact bacteria passage are achieved for multiple gram-negative and gram-positive bacteria. The technique is shown to enable rapid sample preparation and bacteria analysis by single-cell Raman spectrometry. The selective lysis technique presents a unique sample preparation step supporting rapid and culture-free analysis of bacteria for the point of care.