SIZE-BASED CAPTURE OF BACTERIA VIA DENDRITIC FREEZE- CAST CERAMIC MEMBRANES
5.1 Introduction
C h a p t e r 5
SIZE-BASED CAPTURE OF BACTERIA VIA DENDRITIC FREEZE-
Although rapid detection of low concentrations of pathogens in a complex fluid such as blood presents many challenges, recent work in point-of-care diagnostics has addressed part of the problem through the development of digital quantitative detection. Schoepp et al.
developed a digital antimicrobial susceptibility test (dAST) that determined susceptibility of isolates from urinary tract infections in under 15 minutes3. A critical step in dAST was the use of targeted amplification to increase the signal from very low concentrations of pathogen DNA, thereby allowing digital detection. A study by Schlappi et al. used a similar in situ amplification technique in combination with flow-through capture to detect zeptomolar concentrations of pathogen DNA from several milliliters of MES (2-(N- morpholino)ethanesulfonic acid) buffer5. Both studies demonstrated capabilities for rapid detection of pathogens at low concentrations; however, there is still a need to develop a method that will quickly (<30 minutes) isolate and concentrate pathogens from complex solutions into a small volume for later detection. The method must also demonstrate high capture efficiency in order to have a sensitivity comparable to that of blood cultures, which are able to detect 1-30 colony forming units (CFU) of bacteria per mL of blood4.
Figure 5.1: Graph showing the patient survival rate and patients with effective antibiotic therapy as a function of time1
Several different approaches to isolate and concentrate pathogens in blood using size based separations have been discussed in the literature. Work by Ohlsson et al. used acoustophoretic plasma generation from blood to isolate bacteria and then used seed particle trapping enrichment and PCR to concentrate and detect them (Figure 5.2a). The authors reported a processing time of 2 hours, a bacteria capture efficiency of 91%, and a limit of detection of 1000 bacteria/mL6. Although this study demonstrated excellent capture efficiency and fast processing times, it also required a high concentration of bacteria for detection and faced challenges with scalability. A study by Faridi et al. used elasto-inertial microfluidics for size based separation of bacteria from whole blood (Figure 5.2b). They reported a bacteria capture efficiency of 76% & 73% while operating at flow rates of 30 &
60 µL/min respectively7. While this method does successfully isolate bacteria from whole blood, the low flow rates and lack of modularity of their microfluidic system will require more than 30 minutes processing time. Hur et al. demonstrated the selective isolation of large particles and cells using laminar vortices with flow rates at the mL/min scale (Figure 5.2c).
Figure 5.2: Illustration of a) acoustic separation and trapping of bacteria from blood6 (Reproduced with permission), b) particle separation using elasto-inertial particle focusing7 (Reproduced under Creative Commons), and c) hydrodynamic focusing and subsequent trapping of large particles in laminar vortices8 (Reproduced with permission)
They reported a capture efficiency of 10% and 23% for cells with average diameters of 12.4 µm and 20 µm respectively8. While isolating pathogens in side cavities using microvortices is an excellent way to concentrate them, the method outlined by Hur et al. relies upon hydrodynamic forces that scale with the particle diameter to move the particles of interest into the side cavities making it impractical for capturing bacteria (Figure 5.2c). As a result, a more suitable method is needed to concentrate and isolate small pathogens like bacteria.
Here we investigate the efficacy of using dendritic freeze-cast ceramics for diffusion based size-separation. The dendritic morphology consists of a main channel, or primary pore, that has side cavities occurring at regular intervals along the pore wall (Figure 5.3a). Similar to the work by Hur et al., as the fluid flow through the main channel passes over the side cavities it was expected to form microvortices in the side cavities as seen in Figure 5.3b.
These microvortices then facilitate the hydrodynamic trapping of small pathogens that diffuse to the side cavities at a faster rate than the large red blood cells. Capturing pathogens via diffusion is an inherently slow process due to the distance traveled by a diffusing particle scaling as t1/2 and therefore requires slow fluid velocities to achieve high capture efficiency.
Although the slow fluid velocities are at odds with the goal of rapidly isolating pathogens, dendritic freeze-cast ceramics have an advantage in terms of high primary pore density (on the order of 1000s per square centimeter)9. The high pore density allows for a low fluid velocity (3.5 to 70 µm/s) in each pore while achieving a fast total flow rate (10 to 200 µL/min). Furthermore, increasing the total volumetric flow rate while maintaining the same fluid velocity in the primary pores only requires an increase in the area of the ceramic. In this
Figure 5.3: a) SEM micrograph of dendritic ceramic in the longitudinal direction, primary pore outlined in red and side cavities outlined in yellow. b) Illustration of fluid flow through dendritic channels with large red blood cells flowing throw primary pores and small pathogens caught in recirculating flow.
chapter we use a model system comprising different sizes of polystyrene microparticles (PP) to characterize the performance of two dendritic structures. Then, the capability of dendritic ceramics with different surface functionalities was tested using E. coli as model bacteria.
5.2 Experimental Methods