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.

Many biomarker-based diagnostic methods are inhibited by nontarget molecules in patient samples, necessitating biomarker extraction before detection. We have developed a simple device that purifies RNA, DNA, or protein biomarkers from complex biological samples without robotics or fluid pumping. The device design is based on functionalized magnetic beads, which capture biomarkers and remove background biomolecules by magnetically transferring the beads through processing solutions arrayed within small-diameter tubing. The process was automated by wrapping the tubing around a disc-like cassette and rotating it past a magnet using a programmable motor. This device recovered biomarkers at ~80% of the operator-dependent extraction method published previously. The device was validated by extracting biomarkers from a panel of surrogate patient samples containing clinically relevant concentrations of (1) influenza A RNA in nasal swabs, (2) Escherichia coli DNA in urine, (3) Mycobacterium tuberculosis DNA in sputum, and (4) Plasmodium falciparum protein and DNA in blood. The device successfully extracted each biomarker type from samples representing low levels of clinically relevant infectivity (i.e., 7.3 copies/µL of influenza A RNA, 405 copies/µL of E. coli DNA, 0.22 copies/µL of TB DNA, 167 copies/µL of malaria parasite DNA, and 2.7 pM of malaria parasite protein).

Urine samples are increasingly used for diagnosing infections including Escherichia coli, Ebola virus, and Zika virus. However, extraction and concentration of nucleic acid biomarkers from urine is necessary for many molecular detection strategies such as polymerase chain reaction (PCR). Since urine samples typically have large volumes with dilute biomarker concentrations making them prone to false negatives, another impediment for urine-based diagnostics is the establishment of appropriate controls particularly to rule out false negatives. In this study, a mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA target was added to retrospectively collected urine samples from tuberculosis (TB)-infected and TB-uninfected patients to indicate extraction of intact DNA and removal of PCR inhibitors from urine samples. We tested this design on surrogate urine samples, retrospective 1 milliliter (mL) urine samples from patients in Lima, Peru and retrospective 5 mL urine samples from patients in Cape Town, South Africa. Extraction/PCR control DNA was detectable in 97% of clinical samples with no statistically significant differences among groups. Despite the inclusion of this control, there was no difference in the amount of TB IS6110 Tr-DNA detected between TB-infected and TB-uninfected groups except for samples from known HIV-infected patients. We found a increase in TB IS6110 Tr-DNA between TB/HIV co-infected patients compared to TB-uninfected/HIV-infected patients (N=18, p=0.037). The inclusion of an extraction/PCR control DNA to indicate successful DNA extraction and removal of PCR inhibitors should be easily adaptable as a sample preparation control for other acellular sample types.