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Michael J. Pugia

Bio: Michael J. Pugia is an academic researcher from Bayer Corporation. The author has contributed to research in topics: Dipstick & Reagent. The author has an hindex of 15, co-authored 31 publications receiving 722 citations.
Topics: Dipstick, Reagent, Proteinuria, Albuminuria, Albumin

Papers
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Journal ArticleDOI
TL;DR: A dipstick test plus an optical strip reader that can measure urine protein, albumin, and creatinine and calculate the appropriate ratios provides a better screening test for albuminuria or proteinuria than one measuring only albumin or protein.

149 citations

Journal ArticleDOI
TL;DR: An efficient method for enhanced antibody binding has been developed with the covalent immobilization of an organic linker Dithiobissuccinimidylundecanoate on the GNWA surface for their ability to detect bacteria in clinical concentrations.
Abstract: Infectious disease, commonly caused by bacterial pathogens, is now the world's leading cause of premature death and third overall cause behind cardiovascular disease and cancer. Urinary Tract Infection (UTI), caused by E. coli bacteria, is a very common bacterial infection, a majority in women (85%) and may result in severe kidney failure if not detected quickly. Among hundreds of strains the bacteria, E. coli 0157:H7, is emerging as the most aggressive one because of its capability to produce a toxin causing hemolytic uremic syndrome (HUS) resulting in death, especially in children. In the present study, a project has been undertaken for developing a rapid method for UTI detection in very low bacteria concentration, applying current knowledge of nano-technology. Experiments have been designed for the development of biosensors using nano-fabricated structures coated with elements such as gold that have affinity for biomolecules. A biosensor is a device in which a biological sensing element is either intimately connected to or integrated within a transducer. The basic principle for the detection procedure of the infection is partly based on the enzyme-linked immunosorbent assay system. Anti-E. coli antibody-bound Gold Nanowire Arrays (GNWA) prepared on anodized porous alumina template is used for the primary step followed by binding of the bacteria containing specimen. An alkaline phosphatase-conjugated second antibody is then added to the system and the resultant binding determined by both electrochemical and optical measurements. Various kinds of GNWA templates were used in order to determine the one with the best affinity for antibody binding. In addition, an efficient method for enhanced antibody binding has been developed with the covalent immobilization of an organic linker Dithiobissuccinimidylundecanoate (DSU) on the GNWA surface. Studies have also been conducted to optimize the antibody-binding conditions to the linker-attached GNWA surfaces for their ability to detect bacteria in clinical concentrations.

84 citations

Journal ArticleDOI
TL;DR: The pathophysiology of an anti-inflammatory agent termed urinary trypsin inhibitor (uTi) is described, an important anti- inflammatory substance that is present in urine, blood and all organs, and bikunin, a selective inhibitor of serine proteases are described.
Abstract: Inflammation is an important indicator of tissue injury. In the acute form, there is usually accumulation of fluids and plasma components in the affected tissues. Platelet activation and the appearance in blood of abnormally increased numbers of polymorphonucleocytes, lymphocytes, plasma cells and macrophages usually occur. Infectious disorders such as sepsis, meningitis, respiratory infection, urinary tract infection, viral infection, and bacterial infection usually induce an inflammatory response. Chronic inflammation is often associated with diabetes mellitus, acute myocardial infarction, coronary artery disease, kidney diseases, and certain auto-immune disorders, such as rheumatoid arthritis, organ failures and other disorders with an inflammatory component or etiology. The disorder may occur before inflammation is apparent. Markers of inflammation such as C-reactive protein (CRP) and urinary trypsin inhibitors have changed our appraisal of acute events such as myocardial infarction; the infarct may be a response to acute infection and (or) inflammation. We describe here the pathophysiology of an anti-inflammatory agent termed urinary trypsin inhibitor (uTi). It is an important anti-inflammatory substance that is present in urine, blood and all organs. We also describe the anti-inflammatory agent bikunin, a selective inhibitor of serine proteases. The latter are important in modulating inflammatory events and even shutting them down.

79 citations

Journal ArticleDOI
TL;DR: A system that minimized nonspecific binding by the dye through the use of polymethyl vinyl ethers and bis‐(heptapropylene glycol) carbonate showed a greater chemical specificity for albumin when compared to most other proteins.
Abstract: We developed a dye-binding method for albumin in urine based on bis (3',3"-diiodo4'4"-dihydroxy-5'5"-dinitrophenyl)-3,4,5,6-tetrabr omosulfonphthalein (DIDNTB), a dye that has a higher chemical sensitivity and specificity for albumin when compared to two other commonly used dyes. We prepared urine dipsticks with DIDNTB and certain other compounds to prevent "nonspecific" binding to the dipstick matrix. The detection limit for albumin with DIDNTB as the dye is about 10 mg/L. The extent of dye binding to proteins and other compounds was studied using ultracentrifugation and a selectively permeable membrane that permitted the passage of free but not bound dye; we believe this method is superior to photometric titration. The affinity of the dyes for albumin was found to be pH dependent with stronger binding at pH 1.8 than at pH 7.0. At pH 1.8, DIDNTB had a ca.10-fold greater binding coefficient to albumin when compared to the widely used dyes, tetrabromophenol blue (CI 4430-25-5) or bromophenol blue (CI 115-39-9). We developed a system that minimized nonspecific binding by the dye through the use of polymethyl vinyl ethers and bis-(heptapropylene glycol) carbonate. DIDNTB showed a greater chemical specificity for albumin when compared to most other proteins. The new albumin dipsticks are resistant to many potential interferences at substantial concentrations, making the dipsticks suitable to screen for albuminuria.

54 citations

Journal ArticleDOI
TL;DR: The many reports on the association of albuminuria and risk of renal disease recommend that screening should be done for albumin rather than protein, and the cut-off for the albumin dipsticks probably should be set somewhat lower to reduce the number of false negatives and increase the sensitivity of the dipstick.
Abstract: Beginning in 1974, the Japanese Ministry of Health Welfare directed the screening of schoolchildren for proteinuria. We studied their procedure and methods in 6197 school children and also evaluated a new urine dipstick that measures albumin concentrations down to about 10 mg/l and creatinine down to about 300 mg/l. We used specimens from adult in- and outpatients to test the accuracy of the dipsticks. Based on the quantitative results, we set as cutoffs or = "150" mg/l or an albumin of I "30" mg/l indicated increased risk of developing or having a genitourinary disorder. The sensitivity/specificity of the protein dipstick was 95.1%/95.5%, and the same for the albumin dipstick was 83.8%/93.8%. The cut-off for the albumin dipsticks probably should be set somewhat lower to reduce the number of false negatives and increase the sensitivity of the dipstick. When we compared the quantitative albumin to the protein dipsticks with the above cut-offs, we found the sensitivity/specificity to be 79.3%/94.4%, i.e., much like the albumin dipstick results. The many reports on the association of albuminuria and risk of renal disease recommend that screening should be done for albumin rather than protein. Based on the data from the school children, we estimate that a dipstick albumin of "30" mg/l is borderline increased risk, and that a protein dipstick of "150" mg/l is the same. If we call the dipstick "10" mg/l albumin, "30" mg/l albumin and the "150" mg/l protein results "low risk," then we estimate the prevalence of albuminuria in the school children to be about 2.1% and proteinuria to be about 4.3%. Children with these values should have a quantitative test for albumin and protein. We also tested a dipstick for creatinine and found increasing values with increasing age in both genders; the older boys had significantly higher creatinine values than the older girls and younger boys. For the albumin/creatinine ratio, we found 6028 children with a ratio of or = 30 mg/g indicating increased risk. The ratio may be more useful owing to the likely reduction of the number of false negatives and false positives.

32 citations


Cited by
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Journal Article
TL;DR: In the early 1990s, the National Kidney Foundation (K/DOQI) developed a set of clinical practice guidelines to define chronic kidney disease and to classify stages in the progression of kidney disease.

10,265 citations

Journal ArticleDOI
TL;DR: This communication describes a simple method for patterning paper to create well-defined, millimeter-sized channels, comprising hydrophilic paper bounded by hydrophobic polymer, that will become the basis for low-cost, portable, and technically simple multiplexed bioassays.
Abstract: 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.

2,580 citations

Journal ArticleDOI
TL;DR: This review summarizes emerging efforts in combating against infectious diseases, particularly using antimicrobial NPs and antibiotics delivery systems as new tools to tackle the current challenges in treating infectious diseases.

1,493 citations

Journal ArticleDOI
TL;DR: A prototype system for quantifying bioassays and for exchanging the results of the assays digitally with physicians located off-site offers new opportunities for inexpensive monitoring of health, especially in situations that require physicians to travel to patients to obtain diagnostic information that might be obtained more effectively by less valuable personnel.
Abstract: 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...

1,393 citations

Journal ArticleDOI
TL;DR: A method for fabricating 3D microfluidic devices by stacking layers of patterned paper and double-sided adhesive tape to carry out a range of new analytical protocols simply and inexpensively without external pumps is described.
Abstract: 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.

1,204 citations