K/DOQI clinical practice guidelines for chronic kidney disease: Evaluation, classification, and stratification

This communication describes a simple method for patterning paper to create well-defined, millimeter-sized channels, comprising hydrophilic paper bounded by hydrophobic polymer. We believe that this type of patterned paper will become the basis for low-cost, portable, and technically simple multiplexed bioassays. We demonstrate this capability by the simultaneous detection of glucose and protein in 5 μL of urine. The assay system is small, disposable, easy to use (and carry), and requires no external equipment, reagents, or power sources. We believe this kind of system is attractive for uses in less-industrialized countries, in the field, or as an inexpensive alternative to more advanced technologies already used in clinical settings.[1-4]

The analysis of biological fluids is necessary for monitoring the health of populations,[2] but these measurements are difficult to implement in remote regions such as those found in less-industrialized countries, in emergency situations, or in home health-care settings.[3] Conventional laboratory instruments provide quantitative measurements of biological samples, but they are unsuitable for these situations since they are large, expensive, and require trained personnel and considerable volumes of biological samples.[2] Other bioassay platforms provide alternatives to more expensive instruments,[5-7] but the need remains for a platform that uses small volumes of sample and that is sufficiently inexpensive to be used widely for measuring samples from large populations.

We believe that paper may serve as a particularly convenient platform for running bioassays in the remote situations locations. As a prototype for a mthod we believe to be particularly promosing, we patterned photoresist onto chromatography paper to form defined areas of hydrophilic paper separated by hydrophobic lines or “walls”; these patterns provide spatial control of biological fluids and enable fluid transport, without pumping, due to capillary action in the millimeter-sized channels produced. This method for patterning paper makes it possible to run multiple diagnostic assays on one strip of paper, while still using only small volumes of a single sample. In a fully developed technology, patterned photoresist would be replaced by an appropriate printing technology, but patterning paper with photoresist is: i) convenient for prototyping these devices, and ii) a useful new micropatterning technology in its own right.

We patterned chromatography paper with SU-8 2010 photoresist as shown in Figure 1a and as described below: we soaked a 7.5-cm diameter piece of chromatography paper in 2 mL of SU-8 2010 for 30 s, spun it at 2000 rpm for 30 s, and then baked it at 95 °C for 5 min to remove the cyclopentanone in the SU-8 formula. We then exposed the photoresist and paper to 405 nm UV light (50 mW/cm2) for 10 s through a photo-mask (CAD/Art Services, Inc.) that was aligned using a mask aligner (OL-2 Mask Aligner, AB-M, Inc). After exposure, we baked the paper a second time at 95 °C for 5 min to cross-link the exposed portions of the resist. The unpolymerized photoresist was removed by soaking the paper in propylene glycol monomethyl ether acetate (PGMEA) (5 min), and by washing the pattern with propan-2-ol (3 × 10 mL). The paper was more hydrophobic after it was patterned, presumably due to residual resist bound to the paper, so we exposed the entire surface to an oxygen plasma for 10 s at 600 millitorr (SPI Plasma-Prep II, Structure Probe, Inc) to increase the hydrophilicity of the paper (Figures 2a and 2b).



Figure 1

Chromatography paper patterned with photoresist. The darker lines are cured photoresist; the lighter areas are unexposed paper. (a) Patterned paper after absorbing 5 μL of Waterman red ink by capillary action. The central channel absorbs the sample ...





Figure 2

Assays contaminated with (a) dirt, (b) plant pollen, and (c) graphite powder. The pictures were taken before and after running an artificial urine solution that contained 550 mM glucose and 75 μM BSA. The particulates do not move up the channels ...



The patterned paper can be derivatized for biological assays by adding appropriate reagents to the test areas (Figures 1b and ​and2b).2b). In this communication, we demonstrate the method by detecting glucose and protein,[8] but the surface should be suitable for measuring many other analytes as well.[7] The glucose assay is based on the enzymatic oxidation of iodide to iodine,[9] where a color change from clear to brown is associated with the presence of glucose.[10] The protein assay is based on the color change of tetrabromophenol blue (TBPB) when it ionizes and binds to proteins;[11] a positive result in this case is indicated by a color change from yellow to blue.

For the glucose assay, we spotted 0.3 μL of a 0.6 M solution of potassium iodide, followed by 0.3 μL of a 1:5 horseradish peroxidase/glucose oxidase solution (15 units of protein per mL of solution). For the protein assay, we spotted 0.3 μL of a 250-mM citrate buffer (pH 1.8) in a well separate from the glucose assay, and then layered 0.3 μL of a 3.3 mM solution of tetrabromophenol blue (TBPB) in 95% ethanol over the citrate buffer. The spotted reagents were allowed to air dry at room temperature. This pre-loaded paper gave consistent results for the protein assay regardless of storage temperature and time (when stored for 15 d both at 0 °C and at 23 °C, wrapped in aluminum foil). The glucose assay was sensitive to storage conditions, and showed decreased signal for assays run 24 h after spotting the reagents (when stored at 23 °C); when stored at 0 °C, however, the glucose assay was as sensitive after day 15 as it was on day 1.

We measured artificial samples of glucose and protein in clinically relevant ranges (2.5-50 mM for glucose and 0.38-7.5 μM for bovine serum albumin (BSA))[12, 13] by dipping the bottom of each test strip in 5 μL of a pre-made test solution (Figure 2d). The fluid filled the entire pattern within ca. one minute, but the assays required 10-11 min for the paper to dry and for the color to fully develop.[14] In all cases, we observed color changes corresponding roughly in intensity to the amount of glucose and protein in the test samples, where the lowest concentrations define the lower limits to which these assays can be used (Figure 2e). For comparison, commercially-available dipsticks detect glucose at concentrations as low as 5 mM[7, 9] and protein as low as 0.75 μM;[6, 15] these limits indicate that these paper-based assays are comparable in sensitivity to commercial dipstick assays. Our assay format also allows for the measurement of multiple analytes.

This paper-based assay is suitable for measuring multiple samples in parallel and in a relatively short period of time. For example, in one trial, one researcher was able to run 20 different samples (all with 550 mM glucose and 75 μM BSA) within 7.5 min (followed by another 10.5 min for the color to fully develop). An 18-min assay of this type—one capable of measuring two analytes in 20 different sample—may be efficient enough to use in high-throughput screens of larger sample pools.

In the field, samples will not be measured under sterile conditions, and dust and dirt may contaminate the assays. The combination of paper and capillary action provides a mechanism for separating particulates from a biological fluid. As a demonstration, we purposely contaminated the artificial urine samples with quantities of dirt, plant pollen, and graphite powder at levels higher than we might expect to see in the samples in the field. These particulates do not move up the channels under the action of capillary wicking, and do not interfere with the assay (Figure 3).

Paper strips have been used in biomedical assays for decades because they offer an inexpensive platform for colorimetric chemical testing.[1] Patterned paper has characteristics that lead to miniaturized assays that run by capillary action (e.g., without external pumping), with small volumes of fluids. These methods suggest a path for the development of simple, inexpensive, and portable diagnostic assays that may be useful in remote settings, and in particular, in less-industrialized countries where simple assays are becoming increasingly important for detecting disease and monitoring health,[16, 17], for environmental monitoring, in veterinary and agricultural practice and for other applications.

/pdf/patterned-paper-as-a-platform-for-inexpensive-low-volume-1csojtz6lz.pdf

Patterned Paper as a Platform for Inexpensive, Low‐Volume, Portable Bioassays

"Nanoantibiotics": a new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era.

This article describes a prototype system for quantifying bioassays and for exchanging the results of the assays digitally with physicians located off-site. The system uses paper-based microfluidic devices for running multiple assays simultaneously, camera phones or portable scanners for digitizing the intensity of color associated with each colorimetric assay, and established communications infrastructure for transferring the digital information from the assay site to an off-site laboratory for analysis by a trained medical professional; the diagnosis then can be returned directly to the healthcare provider in the field. The microfluidic devices were fabricated in paper using photolithography and were functionalized with reagents for colorimetric assays. The results of the assays were quantified by comparing the intensities of the color developed in each assay with those of calibration curves. An example of this system quantified clinically relevant concentrations of glucose and protein in artificial uri...

/pdf/simple-telemedicine-for-developing-regions-camera-phones-and-4d9clsku42.pdf

Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time, off-site diagnosis.

This article describes a method for fabricating 3D microfluidic devices by stacking layers of patterned paper and double-sided adhesive tape. Paper-based 3D microfluidic devices have capabilities in microfluidics that are difficult to achieve using conventional open-channel microsystems made from glass or polymers. In particular, 3D paper-based devices wick fluids and distribute microliter volumes of samples from single inlet points into arrays of detection zones (with numbers up to thousands). This capability makes it possible to carry out a range of new analytical protocols simply and inexpensively (all on a piece of paper) without external pumps. We demonstrate a prototype 3D device that tests 4 different samples for up to 4 different analytes and displays the results of the assays in a side-by-side configuration for easy comparison. Three-dimensional paper-based microfluidic devices are especially appropriate for use in distributed healthcare in the developing world and in environmental monitoring and water analysis.

/pdf/three-dimensional-microfluidic-devices-fabricated-in-layered-487blmp2vn.pdf

Three-dimensional microfluidic devices fabricated in layered paper and tape.

Detection of low-molecular-weight proteins in urine by dipsticks.

Disclosed is an assay for an analyte in a fluid test sample such as urine which involves combining the fluid test sample with a reagent system comprising an apo-peroxidase, a redox dye, a peroxide and a metal porphyrin which is bound to an analyte/analyte specific binding partner which complex has a combined molecular weight of at least about 180 K Daltons. When this conjugate interacts with analyte in the fluid test sample, a portion of the specific binding partner is dissociated from the complex thereby enabling the metal porphyrin to reconstitute with the apo-peroxidase. The reconstituted peroxidase can interact with the peroxide and redox dye to provide a colored response to analyte in the fluid test sample.

Competitive apo-peroxidase assay

PROBLEM TO BE SOLVED: To enhance correctness of a semiquantitative method by correcting a concentration of a first analysis object in a humor with a concentration of a second analysis object related clinically to the first analysis object. SOLUTION: In the case of albumin in urine in an effective analysis range (30-300 mg/l), a concentration of creatine as a second analysis object is measured, a ratio of albumin/creatine is calculated and a correction concentration of albumin in a sample is obtained. Meanwhile, outside the effective analysis range, the concentration of creatine is not measured, and the ratio of albumin/ creatine is determined with the use of a value of creatine of general healthy people. Errors in estimated values are offset accordingly and a concentration of albumin is determined with more correctness.

Method for improving correctness of semiquantitative determination of analysis object in fluid sample

PROBLEM TO BE SOLVED: To effectuate improved creatinine measurement by bringing a fluid sample into contact with an oxidizing dye showing a color response, measuring the intensity of the color response, and comparing the intensity with an intensity obtained when creatinine of a known concentration is included. SOLUTION: The improved creatinine evaluation requires an evaluation medium including an oxidizing dye generating a color response in the presence of secondary copper ions, hydroperoxide, citric acid and creatinine. A supply source for the secondary copper ions may be any soluble copper salt that does not interact harmfully. The inspection can be carried out by a wet format or dry format. In executing the inspection, a reagent test piece or reagent aqueous solution and an acetonitrile solution are used and mixed with a copper salt, e.g. secondary copper citrate, a dye and hydroperoxide with a pH, preferably 4.0-9.0 whereby the sample is buffered. Limited use of phytin and/or its specific derivative is effective to stabilize a compound for measuring creatinine.

Improved creatinine detection and inspection

The determination of lysozyme in urine is carried out by contacting the urine with a reagent system containing a buffer and a protein error indicator dye. Other proteins normally found in urine can compete with the lysozyme for interaction with the protein error indicator thereby affecting the specificity of the test for lysozyme. The present invention involves the addition of certain alkyl sulfonic acids or sulfonates to increase both specificity and sensitivity of the reagent for lysozyme.

Michael J. Pugia

Papers

Detection of low-molecular-weight proteins in urine by dipsticks.

Competitive apo-peroxidase assay

Method for improving correctness of semiquantitative determination of analysis object in fluid sample

Improved creatinine detection and inspection

Method for the detection of lysozyme using a protein error indicator dye in conjunction with an alkane sulfonic acid