Kenneth R. Hawkins

A rapid diffusion immunoassay in a T-sensor

We have developed a rapid diffusion immunoassay that allows measurement of small molecules down to subnanomolar concentrations in <1 min. This competitive assay is based on measuring the distribution of a labeled probe molecule after it diffuses for a short time from one region into another region containing antigen-specific antibodies. The assay was demonstrated in the T-sensor, a simple microfluidic device that places two fluid streams in contact and allows interdiffusion of their components. The model analyte was phenytoin, a typical small drug molecule. Clinically relevant levels were measured in blood diluted from 10- to 400-fold in buffer containing the labeled antigen. Removal of cells from blood samples was not necessary. This assay compared favorably with fluorescence polarization immunoassay (FPIA) measurements. Numerical simulations agree well with experimental results and provide insight for predicting assay performance and limitations. The assay is homogeneous, requires <1 μl of reagents and sample, and is applicable to a wide range of analytes.

Diffusion based analysis in a sheath flow microchannel: the sheath flow T-sensor

This paper describes a microfluidic channel that allows for diffusion-based analysis of adsorbing species without passivation of the channel surfaces. The sheath flow configuration was used to measure the diffusion coefficient of fluorescently labeled species from their spatial distribution within the microchannel by analyzing the derivative of the intensity profile at the interface between two distinct core fluids. Measurements for both a small molecule (rhodamine B) and an intermediate-sized protein (wheat germ agglutinin) were made, demonstrating the utility of the sheath flow T-sensor.

A method for characterizing adsorption of flowing solutes to microfluidic device surfaces

We present a method for characterizing the adsorption of solutes in microfluidic devices that is sensitive to both long-lived and transient adsorption and can be applied to a variety of realistic device materials, designs, fabrication methods, and operational parameters. We have characterized the adsorption of two highly adsorbing molecules (FITC-labeled bovine serum albumin (BSA) and rhodamine B) and compared these results to two low adsorbing species of similar molecular weights (FITC-labeled dextran and fluorescein). We have also validated our method by demonstrating that two well-known non-fouling strategies [deposition of the polyethylene oxide (PEO)-like surface coating created by radio-frequency glow discharge plasma deposition (RF-GDPD) of tetraethylene glycol dimethyl ether (tetraglyme, CH(3)O(CH(2)CH(2)O)(4)CH(3)), and blocking with unlabeled BSA] eliminate the characteristic BSA adsorption behavior observed otherwise.

Microfluidic diagnostics for low-resource settings

Diagnostics for low-resource settings need to be foremost inexpensive, but also accurate, reliable, rugged and suited to the contexts of the developing world. Diagnostics for global health, based on minimally-instrumented, microfluidicsbased platforms employing low-cost disposables, has become a very active research area recently-thanks, in part, to new funding from the Bill & Melinda Gates Foundation, the National Institutes of Health, and other sources. This has led to a number of interesting prototype devices that are now in advanced development or clinical validation. These devices include disposables and instruments that perform multiplexed PCR-based assays for enteric, febrile, and vaginal diseases, as well as immunoassays for diseases such as malaria, HIV, and various sexually transmitted diseases. More recently, instrument-free diagnostic disposables based on isothermal nucleic-acid amplification have been developed. Regardless of platform, however, the search for truly low-cost manufacturing methods that would enable affordable systems (at volume, in the appropriate context) remains a significant challenge. Here we give an overview of existing platform development efforts, present some original research in this area at PATH, and reiterate a call to action for more.

Instrument-free nucleic acid amplification assays for global health settings

Many infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health care facilities commonly have only basic equipment and health workers have limited training and little ability to maintain equipment and handle reagents.1 Reliable electric power is a common infrastructure shortfall. In this paper, we discuss a practical approach to the design and development of non-instrumented molecular diagnostic tests that exploit the benefits of isothermal amplification strategies. We identify modular instrument-free technologies for sample collection, sample preparation, amplification, heating, and detection. By appropriately selecting and integrating these instrument-free modules, we envision development of an easy to use, infrastructure independent diagnostic test that will enable increased use of highly accurate molecular diagnostics at the point of care in low-resource settings.

Diffusion immunoassay for protein analytes

The diffusion immunoassay, a new microfluidic assay technique, may be an enabling technology for the development of point-of-care immunoassay systems. We have previously described this novel, clinically significant, quantitative, immunoassay technique based on the differences in the diffusivity of an analyte and the complex of that analyte and a specific antibody; it assayed phenytoin in whole blood. In this report, DIAs for two model protein analytes - horseradish peroxidase and IgG - are described. The key parameters in the DIA and how they interact to change the performance of the DIA when proteins are the analytes are discussed. Simulations for several protein DIAs and preliminary results that suggest that the simulations are accurate are presented. Several methods for increasing, the signal are discussed and simulated.

Analytical Devices Based on Transverse Transport in Microchannels

The manipulation of transport transverse to the flow direction has permitted development of microfluidic devices that allow continuous processing of samples. One of these is a rapid competition immunoassay that relies on apparent changes in the diffusivity of antigen due to binding of the antigen to relatively slowly diffusing antibody. It has also been possible to utilize isoelectric focusing to concentrate and separate different types of analytes into separate flowing streams.

Diagnostics to support malaria elimination: Choosing an appropriate biomarker to target the subclinical Plasmodium falciparum transmission reservoir.

As national malaria control programs contemplate their options for shifting tactics and tools to support malaria elimination, it is useful to review the basic characteristics of target analytes commonly used for malaria diagnosis. We identify several epidemiological nuances that impact choice of target analytes for Plasmodium falciparum transmission and we review key concepts such as parasite biomass concentration, circulating parasite density, infectiousness, sequestration, analyte deletions, and persistence. We then define key requirements for the selection of appropriate target analytes that could serve as Plasmodium falciparum proxy indicators of transmission risk. We focus on the intrinsic themes and attributes of malaria biomarkers and deliberately separate those properties from the detection technologies because if a biomarker does not have the appropriate intrinsic properties to meet elimination needs, no amount of heroic engineering can overcome this shortcoming.

Diagnostic Applications of Biomaterials

Medical diagnostics are devices that aid in the diagnosis of a disease or condition. While there are many different classes of diagnostic devices, biomaterials are most often used as components of in vitro diagnostics (IVDs). IVDs generally measure the presence, count or concentration of an analyte in a sample. Analysis is done in vitro (i.e., in a controlled environment outside the body) to minimize noise and potential interference. Common analytes include biomarkers for disease presence and progression, components of pathogens themselves, and xenobiotic substances. This chapter focuses on types of diagnostics that frequently incorporate biomaterials, and especially on devices that are used in lower-throughput, point-of-care (POC) settings.