A Rapid and Low-Cost Pathogen Detection Platform by Using a Molecular Agglutination Assay.

Rapid and low-cost pathogen diagnostic approaches are critical for clinical decision-making procedures. Cultivating bacteria often takes days to identify pathogens and provide antimicrobial susceptibilities. The delay in diagnosis may result in compromised treatment and inappropriate antibiotic use. Over the past decades, molecular-based techniques have significantly shortened pathogen identification turnaround time with high accuracy. However, these assays often use complex fluorescent labeling and nucleic acid amplification processes, which limit their use in resource-limited settings. In this work, we demonstrate a wash-free molecular agglutination assay with a straightforward mixing and incubation step that significantly simplifies procedures of molecular testing. By targeting the 16S rRNA gene of pathogens, we perform a rapid pathogen identification within 30 min on a dark-field imaging microfluidic cytometry platform. The dark-field images with low background noise can be obtained using a narrow beam scanning technique with off-the-shelf complementary metal oxide semiconductor (CMOS) imagers such as smartphone cameras. We utilize a machine learning algorithm to deconvolute topological features of agglutinated clusters and thus quantify the abundance of bacteria. Consequently, we unambiguously distinguish Escherichia coli positive from other E. coli negative among 50 clinical urinary tract infection samples with 96% sensitivity and 100% specificity. Furthermore, we also apply this quantitative detection approach to achieve rapid antimicrobial susceptibility testing within 3 h. This work exhibits easy-to-use protocols, high sensitivity, and short turnaround time for point-of-care testing uses.

Optofluidic device for label-free cell classification from whole blood.

A unique optofluidic lab-on-a-chip device that can detect optically encoded forward scattering signals is demonstrated. With a unique design of a spatial mask that patterns the intensity distribution of the illuminating light, the position and velocity of each travelling cell in the flow can be measured with submicrometer resolution, which enables the generation of a cell distribution plot over the cross section of the channel. The distribution of cells is highly sensitive to its size and stiffness, both being important biomarkers for cell classification without cell labelling. The optical-coding technique offers an easy route to classify cells based on their size and stiffness. Because the stiffness and size of neutrophils are distinct from other types of white blood cells, the number of neutrophils can be detected from other white blood cells and red blood cells. Above all, the enumeration of neutrophil concentration can be obtained from only 5 µL of human blood with a simple blood preparation process saving the usual steps of anticoagulation, centrifugation, antibody labelling, or filtering. The optofluidic system is compact, inexpensive, and simple to fabricate and operate. The system uses a commodity laser diode and a Si PIN photoreceiver and digital signal processing to extract vital information about cells and suppress the noise from the encoded optical scattering signals. The optofluidic device holds promise to be a point-of-care and home care device to measure neutrophil concentration, which is the key indicator of the immune functions for cancer patients undergoing chemotherapy.

Counting leukocytes from whole blood using a lab-on-a-chip Coulter counter.

A microfluidic lab-on-a-chip Coulter counter was demonstrated to count micro particles and leukocytes from whole blood. Instead of electroplated or deposited metal electrodes, off-the-shelf gold pins were used as electrodes to simplify fabrication process, reduce cost, enhance device durability, and above all, achieve superior uniformity in E-field distribution for improved signal quality. A custom-designed, low-cost demodulation circuit was developed to detect the AC impedance signals of the particles and cells passing the detection area defined by the microfluidic channels. A mixture of polystyrene beads with three different sizes was used to characterize the device. The results showed high throughput at 2000 particles/s and clear separation among different sizes of beads with coefficients of variation (CV) of 13.53%, 10.35% and 5.67% for 7.66 µm, 10.5 µm and 14.7 µmbeads, respectively. To demonstrate the potential for a point-of-care or self-administered device for cancer patients going through chemotherapy, we have used the lab-on-a-chip device to count leukocytes from whole blood, generating encouraging preliminary results comparable to the results from a commercial flow cytometer.