Biosensors are important tools for measuring concentrations of specific analytes found in biological fluids. Paper-based microfluidic devices can offer many benefits to the field of biosensor development. Paper-based materials are inexpensive, easily disposable, and readily available. Small volumes of fluid can be used in these devices, which decrease the need for costly reagents. And capillary flow in paper materials removes the need for pumping equipment. The topic of this presentation is a paper-based, biosensing device that can be used for therapeutic, home monitoring by people with the genetic disorder Phenylketonuria (PKU.)
People affected by PKU are required to maintain a strict, low Phenylalanine (Phe) diet to avoid irreversible, adverse health affects. Maintaining appropriate levels of Phe can be especially challenging for young children, adolescents and pregnant women. Much like a glucose test kit helps a person with diabetes maintain appropriate glucose levels, a device that can monitor the concentration of Phe in a finger prick of blood could aid a person with PKU maintain appropriate Phe levels and adhere to diet therapy. Current testing for Phe is laboratory-based, requiring expensive equipment or trained technicians.
Our proposed solution is a semi-quantitative, paper-based assay that utilizes a well-known, two-step chemical reaction to produce a colorimetric response that correlates Phe concentration to color intensity. The assay accepts a finger prick volume of blood (40 μL) into an asymmetric polymeric membrane that filters out cellular components and allows plasma to wick into two glass fiber pads where the first reaction takes place for six minutes. These pads have been impregnated with Phenylalanine Dehydrogenase (PheDH) and the oxidized form of Nicotinamide adenine dinucleotide (NAD+.) Phe is catalyzed by PheDH and the reaction reduces NAD+ into NADH. A third glass fiber pad, impregnated with Nitro blue tetrazolium chloride (NBT) and methoxy Phenazine Methyl Sulfate (mPMS,) is then folded to make contact with a pad containing the products of the first reaction to allow for a second reaction to take place for an additional minute. NBT is reduced by NADH from the first reaction (in a process aided by mPMS) to its formazan dye product. This visible color change is recorded with a desktop scanner. Statistically relevant intensity differences have been measured in concentrations of blood spiked with 0 (normal), 3.75 (slightly elevated) and >7.5 (substantially elevated) mg/dL Phe using this device.
The focus of this presentation will be on optimization of the flow path of reagents to achieve maximum signal-to-noise ratio in the detection region by controlling the patterning and drying of reagents in the multi-component assay.