DISTANCE-BASED QUANTITATIVE ANALYSIS USING A CAPILLARITY-BASED ANALYTICAL DEVICE

Apparatus for quantitative analytical measurements using capillarity-based analytical devices is described. Porous cellulose (i.e., common filter paper) may be used as the reagent carrier for the analyses. Hydrophobic materials may be printed onto the paper to generate paths that restrict liquid flow by capillary action to defined regions. At least one colorimetric reagents effective for reacting with a specific analyte is deposited along a capillary flow path generated in the device. Upon placing the liquid containing the analyte on one end of the path, the liquid moves along the circuit by capillary action, and the flowing analyte reacts with reagent generating color along the flow path until all of the analyte is consumed. Analyte quantification is achieved by measuring the length of the colored portion along a flow path employing a direct-reading measurement scale. Analyses are demonstrated for chemical detection modalities including: enzymatic action, metal complexation, and nanoparticle aggregation, but the present apparatus may be used for a wide range of analytes.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention include a simple apparatus for quantitative, capillarity-based analyses having broad chemical applicability (See, “Simple, Distance-Based Detection for Paper Analytical Devices,” by David M. Cate et al., Lab on a Chip 13 (12): 2397-2404 (25 April 2013) doi:10.1039/C3LC50072A which is hereby incorporated by reference herein for all that it discloses and teaches.). Hydrophobic materials may be printed onto the paper for defining flow circuits or paths that restrict liquid flow by capillary action to defined regions. At least one colorimetric reagent effective for reacting with a specific analyte is deposited along a capillary flow path generated in the capillarity-based device. Upon placing the liquid containing the analyte on one end of the circuit, the liquid moves along the path by capillary action, whereby as the flowing analyte reacts with reagent, color develops along the flow path until all of the analyte is consumed. Analyte quantification is achieved by measuring the length of the colored portion along the flow path, using a direct-reading measurement scale formed alongside or on the flow path, thus eliminating the need to differentiate color hues and intensities by the user as is typical with existing PADs. Assays based on color length were developed that use enzymatic action, metal complexation, and nanoparticle aggregation. Each assay provided quantitative detection of different analytes within specific biological and environmental matrices of interest.

Reference will now be made in detail to the present embodiments of the invention, examples of which are illustrated in the accompanying drawings. In the FIGURES, similar structure will be identified using identical reference characters. It will be understood that the FIGURES are for the purpose of describing particular embodiments of the invention and are not intended to limit the invention thereto. Turning now toFIG. 1, an embodiment of the capillarity-based analytic device,10, of the present invention is illustrated, whereFIG. 1Ais a schematic representation of a top view of an embodiment of elongated substrate,12, illustrating a liquid-confining path,14, formed thereon having a first end,16, and a second end,18, into which colorimetric reagents effective for reacting with a specific analyte are deposited, and fluid well,20, formed near first end,16, thereof. A wax ink may be designed and printed onto substrate12, using graphics software, and subsequently heated to generate a two-dimensional  liquid-confining channel, the top and bottom confinement being generated using liquid-impervious sheeting, as will be described in more detail hereinbelow. The substrate used for the analyses set forth in the EXAMPLES hereinbelow was standard cellulosic filter paper. However, any porous hydrophilic material that can be patterned or cut into the desired shape may be used for such assays. Other examples include, glass, nitrocellulose, silk, and cotton. For non-aqueous, non-polar systems, hydrophobic substrates such as nylon, Polytetrafluoroethylene (PTFE), Polyvinylidene fluoride (PVDF), or other halogenated polymers capable of providing sufficient chemical resistance and effective for wicking non-polar organic solvents, may be used. Colorimetric detection reagents were deposited along the flow channel by spray application or by use of a pipette, as examples. For spray application, a nebulizer is used to deposit reagent droplets uniformly along the channel. This process is rapid, but inefficient, since significant amounts of reagent are deposited onto the surrounding paper; that is, outside of the flow circuit. Although these reagents do not affect the assay results because they are separated from the flow channel by the wax barrier, they are wasteful. Alternatively, a pipette was used to deposit the reagents onto the paper in minute (approximately 0.5 μL, as an example) increments, which provides more efficient use of reagents. Once the deposited reagents are dry, device10is ready for use.

FIG. 1Bis a schematic representation of a top view of assembled device10showing direct-reading measuring scale,22, imprinted either on the surface of assembled device10or on substrate12, and orifice,24, in top liquid impervious surface,26, permitting access to liquid well20of substrate12, for sample addition. Substrate12below orifice24may be retained for holding reagents for sample pre-treatment, or removed to facilitate sample transfer into the detection zone. As stated hereinabove, a liquid sample is introduced into the sample reservoir and then carried by capillary action along the flow channel. As the analyte reacts with its reagent, a colored product develops. Once all of the analyte has reacted, the color development stops (even though the solution continues to flow along the channel), the developed colored product remaining where it was generated. Analyte quantification is then performed by measuring the length of the colored region on the flow channel using the direct-reading measurement scale. Samples were introduced into the sample well using a syringe. Other methods of sample introduction include: (a) dipping the well portion of the device directly into a liquid solution containing the analyte; (b) using inertial impaction to deposit airborne particulate matter into the well and solubilizing the particulate matter using a suitable liquid for dissolving and carrying the dissolved material into the channel; and (c) flowing gas (or liquid) through the sample addition well orthogonal to the capillarity flow path, assuming there was no backing/laminate. Some portion of the analyte in the reservoir could then be trapped or sequestered, or the orthogonal flow is allowed to migrate into the analysis channel, thereby introducing analyte into the channel.

FIG. 1Cis a schematic representation of a side view of the assembled apparatus, illustrating substrate12having liquid impervious layers,26, and,28, on both sides thereof.FIG. 1Dis a perspective view of the assembled device10illustrated inFIG. 1B, showing an expanded view of orifice24thereof.

FIG. 2Ais a schematic representation of a top view of a second embodiment of elongated substrate12of the capillarity-based analytic device10hereof. In this embodiment, the liquid-confining path is formed by substrate12, itself, when sandwiched between two liquid-impervious sheets, there being no requirement to use a wax ink as described hereinabove. Colorimetric reagents effective for reacting with a specific analyte are deposited onto substrate12before it is covered. Liquid well24is formed near end20thereof.FIG. 2Bis a schematic representation of a top view of assembled device10, showing direct-reading measuring scale22imprinted on liquid-impervious surface26of assembled device10, and orifice24in top, liquid-impervious surface26, whereby liquid is permitted access to liquid well20of substrate12.FIG. 2Cis a schematic representation of a side view of the assembled apparatus, illustrating substrate12shown inFIG. 2Ahereof enclosed by liquid impervious layers26and28.FIG. 2Dis a perspective view of assembled device10illustrated inFIG. 2B, hereof showing an expanded view of orifice24thereof.

FIG. 3Ais a schematic representation of a top view of an embodiment of elongated substrate12of capillarity-based analytic device10hereof, illustrating liquid-confining path14formed therethrough in a similar manner to that described forFIG. 1Ahereof, except that the liquid confining path is not linear, but provides a more circuitous route along the substrate for situations where the reaction kinetics are slow. As an example, wax baffles,30, and,32, may be printed on substrate12, and used to divert liquid flow in a nonlinear manner. Colorimetric reagents effective for reacting with the analyte are again deposited, and liquid well20is formed near end16thereof.FIG. 3Bis a schematic representation of a top view of assembled device10, showing scale22imprinted either on liquid impermeable surface26of device10, or on substrate12, and orifice24in top liquid impermeable surface26, thereby permitting fluid access to liquid well20of substrate12.FIG. 3Cis a schematic representation of a side view of the assembled apparatus, illustrating substrate12having liquid impervious layers28and30on either side thereof.FIG. 3Dis a perspective view of assembled device10illustrated inFIG. 3B, showing an expanded view of orifice24thereof.

FIG. 4illustrates fabrication and assembly of the embodiment of the device shown inFIGS. 1A-1D, hereof. After a wax ink is printed onto substrate12, and subsequently heated to generate a two-dimensional liquid-confining channel14, step34, shows the deposition of colorimetric reagents effective for reacting with a specific analyte, by spray application or by pipetting, as examples. The reagents are then allowed to dry. Step36illustrates the placement of transparent, liquid-impervious sheet26having measuring scale22printed thereon and having hole or orifice24therein to permit liquid access to liquid well,20, formed near first end,16, thereof, onto substrate12, and the placement of second liquid impervious sheet28, which may not be transparent, onto the bottom of substrate12. Step38seals sheets26and28to substrate12, using a thermal laminating process, as an example, as is known in the art, completing measuring apparatus10. The sealing of sheets28and30to substrate12completes the formation of liquid confinement channel. Clearly, other methods for creating a liquid impermeable barrier on substrate12are envisioned, one being a coating process.

FIG. 5illustrates fabrication and assembly of the embodiment of the device shown inFIGS. 2A-2D, hereof, andFIG. 6illustrates fabrication and assembly of the embodiment of the device shown inFIGS. 3A-3D, hereof, by similar process steps to those shown inFIG. 3.

FIG. 7Ais a graph of the distance in millimeters of color development in the apparatus illustrated inFIG. 1hereof, as a Log function of a known quantity of analyte in nmols for a glucose analysis system,FIG. 7Bas a Log function of a known quantity of analyte in nmols for a glutathione analysis system, andFIG. 7Cas a function of a known quantity of analyte in nmols for a nickel analysis system, all within the linear range of the reaction, the error bars representing one standard deviation, and the diagrams of the complete reaction are included for each calibration data point. Glucose was detected using glucose oxidase, 3,3′-diaminobenzidine (DAB) and peroxidase, where the glucose oxidase produces hydrogen peroxide that further reacts with DAB in the presence of peroxidase to form a brown, insoluble product (polyDAB). Like DMG, DAB is colorless, but forms a highly colored and easily visualized product in the presence of the analyte. Glutathione (GSH) was detected using a silver nanoparticle (AgNP) aggregation assay, where the AgNPs aggregate in the presence of GSH to form a reddish-brown product that is distinguished from the orange color of the AgNPs in the absence of glutathione. Nickel, as Ni2+, was detected using dimethylglyoxime (DMG) as an example assay for heavy metals, where DMG is placed in the channel and reacts with Ni2+to form a pink product. Solutions containing Ni2+are colorless in the absence of DMG. These reactions will be described in more detail in the EXAMPLES set forth hereinbelow.

Capillarity-based analytical devices have great potential for application at the point-of-need. The quantitative analytical device of embodiments of the present invention is minimally instrumented for device portability, and is highly cost effective; excluding fabrication equipment, a single assay costs approximately $0.04. Since analyte quantification is immediate and can be performed on-site, processing time is significantly reduced when compared to other centralized measurement techniques, which often sacrifice processing speed for detection sensitivity. Like most PAD technologies, however, embodiments of the present invention sacrifice dynamic range for cost, speed, and ease of use. This limitation on reaction stoichiometries can be accommodated in part by tuning the capillarity-based analytical devices hereof to detect different analyte concentration ranges by adjusting reagent concentrations in the flow channel.

Having generally described the invention, the following EXAMPLES provide greater detail. In what follows, cellulosic filter paper was used as the substrate.

Human control serum samples (levels I and II) for both GSH and glucose were obtained from commercial sources. Levels of analytes were provided by the suppliers. Before analysis, unwanted protein was removed from samples using a filter (10 kDa MWCO) and centrifuging for 20 min. at 10,000 rpm for glucose and 10 min. for GSH. In addition, a solution of 5% 5-sulfosaliccylic acid was added prior to centrifugation for GSH.

The capillarity-based paper-based assay for glucose detection consisted of a wax-printed circular reservoir (5 mm diameter) for glucose oxidase (GOD) and peroxidase Type I (HRP) enzyme modification, and a straight channel (2 mm×40 mm) for measuring glucose reaction with peroxidase and DAB. Aliquots (˜0.5 μL) of 600 U/mL glucose oxidase and 500 U/mL HRP were spotted on the sample reservoir and ˜0.5 μL of DAB was pipetted onto the straight channel every five millimeters to account for reagent spreading along the channel length. For each assay, ˜20 μL of the standard or sample solution was added to the sample reservoir. The length of the colored range was found to be proportional to the amount of glucose added over the range of ˜7 nmol to ˜200 nmol. Method variability was relatively low as seen by the small error bars (representing standard deviations of repeat measures) around each datum as illustrated inFIG. 7A. Commercially-available control serum samples known to contain either normal or abnormal glucose levels were also analyzed. Glucose concentrations within the control serum samples are shown inFIG. 7Aas open squares; their alignment with the calibration curve shows the ability of this method to measure glucose accurately and precisely in a relatively complex sample matrix.

The paper assay for glutathione detection consisted of a circular reservoir for sample addition (6 mm diameter) and a baffled flow channel (3 mm×60 mm) divided into 14 equal sections (0.3 mm×2 mm). Flow baffles were used to decrease the capillary flow velocity along the channel, thereby maximizing reaction time between glutathione and the AgNPs. The AgNP solution (˜0.5 μL) was spotted onto each of the 14 sections along the channel. For each assay, ˜20 μL of sample solution was added to the sample reservoir. Complete sample analysis took approximately10min. Assay selectivity was investigated by addition of ˜20 μL of standard thiol solution (˜0.5 nmol), which did not form a colored reaction product along the paper channel.

The spotted detection reagent, AgNP (˜11 nm diameter) turned a dark orange color. The nanoparticles aggregate in the presence of glutathione, which causes a color change from orange to deep red on the paper substrate. A color change from orange to light orange was observed when buffer was added, but was easily distinguished from the dark red of the glutathione-specific product. Detection of glutathione was log-linear for the concentration range tested (˜0.12 nmol to ˜2.0 nmol). The assay selectivity against other thiols (cysteine and homocysteine) and disulfides (cysteine, homocystine, and glutathione disulfide) was also determined. Cysteine and homocysteine were found to cause similar color changes, but the length of color development was much less than for glutathione. None of the disulfides tested caused any color change. The ability to measure glutathione spiked in serum samples (open squares onFIG. 7B) was determined. The measured distances in serum (˜4.2 and ˜5.7 mm) agree well with those of the standard solutions (˜3.7 and ˜5.3 mm) for glutathione concentrations ˜0.25 and ˜0.5 nmol respectively.

A nebulizer was used to saturate the paper surface with DMG (˜50 mM). The deposited reagents were then air dried. The paper was uniformly coated with ammonium hydroxide (pH 9.5), because the rate and extent of Ni2+-DMG complexation are pH dependent, with the fastest rate occurring at a pH of9. To prevent user contamination and excess solvent evaporation, the filter paper was passed through a desktop laminator at 300° F. twice on each side. Laminating the paper also provided better mechanical stability for assay handling. A ˜6.4 mm (ID) hole was punched through the sample reservoir and masking tape was applied to one side to prevent sample loss from leakage during use. For analysis, ˜20 μL of a Ni standard solution (1000 ppm) was deposited onto the sample reservoir. The Ni-DMG complex is reddish pink, precipitates upon formation, and was readily distinguished from the clear sample solution. Color development is rapid and total sample analysis was performed in less than ten minutes. The reaction distance was measured using the naked eye and verified using a desktop scanner. It was found that as the amount of DMG increases, the sensitivity of the assay increases. The assay detection limits are sufficiently low that nmol levels of Ni2+can be detected in the presence of other transition and heavy metals. To measure Ni concentration, the incineration ash was first dissolved in acid and then treated to complex interfering metals. Various dilutions of the resulting solution were analyzed, and the results shown as open squares inFIG. 7C.

An incineration ash sample was purchased for assay validation. Incineration ash along with ˜1 mL concentrated nitric acid was heated in a 20 mL scintillation vial for five min. at ˜250° C. on a hotplate until complete acid evaporation. An ˜262 μL solution containing deionized water (˜250 μL), sodium fluoride, acetic acid (2:1:1 v/v %), and ˜12 μL sodium hydroxide (12 M) was added to the vial. After homogenous mixing with a pipette for several seconds, the solution was centrifuged for 10 min. at 14,000 RPM. For each assay, ˜20 μL of the supernatant was added to the sample reservoir. Good agreement was obtained between measured and known Ni concentrations.