Microfluidic Point-of-Care Ecarin-Based Clotting and Chromogenic Assays for Monitoring Direct Thrombin Inhibitors.

Direct thrombin inhibitors (DTIs), such as bivalirudin and dabigatran, have maintained steady inpatient and outpatient use as substitutes for heparin and warfarin, respectively, because of their high bioavailability and relatively safe "on-therapy" range. Current clinical methods lack the capacity to directly quantify plasma DTI concentrations across wide ranges. At present, the gold standard is the ecarin clotting time (ECT), where ecarin maximizes thrombin activity and clotting time is evaluated to assess DTIs' anticoagulation capability. This work focused on the development of a microfluidic paper analytic device (µPAD) that can quantify the extent of anticoagulation as well as DTI concentration within a patient's whole blood sample. Capillary action propels a small blood sample to flow through the nitrocellulose paper channels. Digital images of whole blood migration are then captured by our self-coded Raspberry Pi and/or the Samsung Galaxy S8 smartphone camera. Both the flow length and the blue absorbance from the plasma front on the µPAD were measured, allowing simultaneous, dual assays: ecarin clotting test (ECT) and ecarin chromogenic assay (ECA). Statistically significant (p < .05) changes in flow and absorbance were observed within our translational research study. Currently, there are no quantitative, commercially available point-of-care tests for the ECT and ECA within the United States. Both the ECT and ECA assays could be instrumental to differentiate between supratherapeutic and subtherapeutic incidents during bridging anticoagulant therapy and limit the unwarranted use of reversal agents.

Flow Rate and Raspberry Pi-based Paper Microfluidic Blood Coagulation Assay Device.

Monitoring blood coagulation in response to an anticoagulant (heparin) and its reversal agent (protamine) is essential during and after surgery, especially with cardiopulmonary bypass (CPB). A current clinical standard is the use of activated clotting time (ACT), where the mechanical movement of a plunger through a whole blood-filled channel is monitored to evaluate the endpoint time of coagulation. As a rapid, simple, low-volume, and cost-effective alternative, we have developed a paper microfluidic assay and Raspberry Pi-based device with the aim of quantifying the extent of blood coagulation in response to varying doses of heparin and protamine. The flow rate of blood through the paper microfluidic channel is automatically monitored using Python-coded edge detection algorithm. For each set of assay, 8 µL of fresh human whole blood (untreated and undiluted) from human subjects is loaded onto each of 8 sample pads, which have been preloaded with varying amounts of heparin or protamine. Total assay time is 3-5 minutes including the time for sample loading and incubation.

Mie scatter spectra-based device for instant, contact-free, and specific diagnosis of bacterial skin infection.

Rapid and specific diagnostic techniques are needed to expedite specific treatment of bacterial skin infections with narrow-spectrum antibiotics, rather than broad-spectrum. Through this work a device was developed to determine the presence of and species responsible for a bacterial skin infection using differences in Mie scatter spectra created by different bacterial species. A 650 nm LED at five different incident angles is used to illuminate the tissue, with Mie scatter being detected by PIN photodiodes at eight different detection angles. Mie scatter patterns are collected at all photodiode angles for each of the incident light angles, resulting in a Mie scatter spectra. Detectable differences in Mie scatter spectra were found using the device developed between commensal bacteria (no infection) and bacteria inoculated (infection) on the surface of both porcine and human cadaveric epidermis. Detectable differences were found between species of infection, specifically Escherichia coli and Staphylococcus aureus, with differences summarized through principle component analysis. Mie scatter spectra can be detected within a few seconds without skin contact. This device is the first to rapidly and specifically diagnose bacterial skin infections in a contact-less manner, allowing for initial treatment with narrow spectrum antibiotics, and helping to reduce the likelihood of resistance.

Angular photodiode array-based device to detect bacterial pathogens in a wound model.

We have developed a device that is able to rapidly and specifically diagnose bacterial pathogens in a wound model based on Mie scatter spectra from a tissue surface. The Mie scatter spectra collected is defined as the intensity of Mie scatter over the angle of detection from a tissue surface. A 650 nm LED perpendicular to the surface illuminates a tissue sample (90°) and photodiodes positioned in 10° increments from 10° to 80° of backscatter act as the detectors to collect these Mie scatter spectra. Through principal component analysis of the Mie scatter spectra collected, we have shown significant differences between Mie scatter spectra of tissues with bacterial pathogens versus those without, as well as significant differences between each species of bacteria tested. The device developed has been tested with a porcine dermis wound model, with samples inoculated with one of three bacterial species (Staphylococcus aureus, Escherichia coli, or Salmonella Typhimurium). Such a device could be critical in the monitoring of a wound for infection and rapid, specific diagnosis of a bacterial wound infection, which would significantly reduce the time and cost associated with specific diagnosis of a bacterial wound infection currently.