The ring pattern resulting from the unique microfluidics in an evaporating coffee drop is a well-studied mass transport phenomenon generating interest in the research community mostly from a mechanistic perspective. In this report, we describe how biomarker-induced particle-particle assemblies, magnetic separation, and evaporation-driven ring formation can be combined for simple pathogen detection. In this assay design, the presence of biomarkers causes self-assembly of a magnetic nanoparticle and a fluorescently labeled micrometer-sized particle. A small spherical magnet under the center of the drop prevents these assemblies from migrating to the drop's edge while a nonreactive control particle flows to the edge forming a ring pattern. Thus the presence or absence of biomarker results in distinctly different distributions of particles in the dried drop. Proof-of-principle studies using poly-L-histidine, a peptide mimic of the malaria biomarker pfHRPII, show that the predicted particle distributions occur with a limit of detection of approximately 200-300 nM.

Effective point-of-care diagnostics require a biomarker detection strategy that is low-cost and simple-to-use while achieving a clinically relevant limit of detection. Here we report a biosensor that uses secondary flows arising from surface Marangoni stresses in an evaporating drop to concentrate target-mediated particle aggregates in a visually detectable spot. The spot size increases with increasing target concentration within the dynamic range of the assay. The particle deposition patterns are visually detectable and easily measured with simple optical techniques. We use optical coherence tomography to characterize the effect of cross-sectional flow fields on the motion of particles in the presence and absence of target (aggregated and non-aggregated particles, respectively). We show that choice of substrate material and the presence of salts and glycerol in solution promote the Marangoni-induced flows that are necessary to produce signal in the proposed design. These evaporation-driven flows generate signal in the assay on a PDMS substrate but not substrates with greater thermal conductivity like indium tin oxide-coated glass. In this proof-of-concept design we use the M13K07 bacteriophage as a model target and 1 μm-diameter particles surface functionalized with anti-M13 monoclonal antibodies. Using standard microscopy-based techniques to measure the final spot size, the assay has a calculated limit-of-detection of approximately 100 fM. Approximately 80% of the maximum signal is generated within 10 minutes of depositing a 1 μL drop of reacted sample on PDMS enabling a relatively quick time-to-result.

The lack of an effective technique for three-dimensional flow visualization has limited experimental exploration of the “coffee ring effect” to the two-dimensional, top-down viewpoint. In this report, high-speed, cross-sectional imaging of the flow fields was obtained using optical coherence tomography to track particle motion in an evaporating colloidal water drop. This approach enables z-dimensional mapping of primary and secondary flow fields and changes in these fields over time. These sectional images show that 1 μm diameter polystyrene particles have a highly nonuniform vertical distribution with particles accumulating at both the air–water interface and the water–glass interface during drop evaporation. Particle density and relative humidity are shown to influence interfacial entrapment, which suggests that both sedimentation rate and evaporation rate affect the dynamic changes in the cross-sectional distribution of particles. Furthermore, entrapment at the air–water interface delays the time at which particles reach the ring structure. These results suggest that the organization of the ring structure can be controlled based on the ratio of different density particles in a colloidal solution.

We report a novel, low-resource malaria diagnostic platform inspired by the coffee ring phenomenon, selective for Plasmodium falciparum histidine-rich protein-II (PfHRP-II), a biomarker indicative of the P. falciparum parasite strain. In this diagnostic design, a recombinant HRP-II (rcHRP-II) biomarker is sandwiched between 1 μm Ni(II)nitrilotriacetic acid (NTA) gold-plated polystyrene microspheres (AuPS) and Ni(II)NTA-functionalized glass. After rcHRP-II malaria biomarkers had reacted with Ni(II)NTA-functionalized particles, a 1 μL volume of the particle-protein conjugate solution is deposited onto a functionalized glass slide. Drop evaporation produces the radial flow characteristic of coffee ring formation, and particle-protein conjugates are transported toward the drop edge, where, in the presence of rcHRP-II, particles bind to the Ni(II)NTA-functionalized glass surface. After evaporation, a wash with deionized water removes nonspecifically bound materials while maintaining the integrity of the surface-coupled ring produced by the presence of the protein biomarker. The dynamic range of this design was found to span 3 orders of magnitude, and rings are visible with the naked eye at protein concentrations as low as 10 pM, 1 order of magnitude below the 100 pM PfHRP-II threshold recommended by the World Health Organization. Key enabling features of this design are the inert and robust gold nanoshell to reduce nonspecific interactions on the particle surface, inclusion of a water wash step after drop evaporation to reduce nonspecific binding to the glass, a large diameter particle to project a large two-dimensional viewable area after ring formation, and a low particle density to favor radial flow toward the drop edge and reduce vertical settling to the glass surface in the center of the drop. This robust, antibody-free assay offers a simple user interface and clinically relevant limits of biomarker detection, two critical features required for low-resource malaria detection.

Atherosclerosis, a leading cause of morbidity and mortality worldwide, is characterized by the accumulation of lipid deposits inside arterial walls, leading to narrowing of the arterial lumen. A significant challenge in the development of diagnostic and therapeutic strategies is to elucidate the contribution of the various cellular participants, including macrophages, endothelial cells, and smooth muscle cells, in the initiation and progression of the atheroma. This protocol details a strategy using quantum dot nanocrystals to monitor homing and distribution of cell populations within atherosclerotic lesions with high signal to noise ratios over prolonged periods of analysis. This fluorescence-based approach enables the loading of quantum dots into cells such as macrophages without perturbing native cell functions in vivo, and has been used for the multiplexed imaging of quantum dot-labeled cells with biomarkers of atherosclerotic disease using conventional immunofluorescence techniques.