Development of a Quantitative Digital Urinalysis Tool for Detection of Nitrite, Protein, Creatinine, and pH.

This paper presents a cost-effective, quantitative, point-of-care solution for urinalysis screening, specifically targeting nitrite, protein, creatinine, and pH in urine samples. Detecting nitrite is crucial for the early identification of urinary tract infections (UTIs), while regularly measuring urinary protein-to-creatinine (UPC) ratios aids in managing kidney health. To address these needs, we developed a portable, transmission-based colorimeter using readily available components, controllable via a smartphone application through Bluetooth. Multiple colorimetric detection strategies for each analyte were identified and tested for sensitivity, specificity, and stability in a salt buffer, artificial urine, and human urine. The colorimeter successfully detected all analytes within their clinically relevant ranges: nitrite (6.25-200 µM), protein (2-1024 mg/dL), creatinine (2-1024 mg/dL), and pH (5.0-8.0). The introduction of quantitative protein and creatinine detection, and a calculated urinary protein-to-creatinine (UPC) ratio at the point-of-care, represents a significant advancement, allowing patients with proteinuria to monitor their condition without frequent lab visits. Furthermore, the colorimeter provides versatile data storage options, facilitating local storage on mobile devices or in the cloud. The paper further details the setup of the colorimeter's secure connection to a cloud-based environment, and the visualization of time-series analyte measurements in a web-based dashboard.

Toward a Quantitative Colorimeter for Point-of-Care Nitrite Detection.

This paper reports on a low-cost, quantitative, point-of-care solution for the early detection of nitrite, a common biomarker for urinary tract infections (UTIs). In a healthy individual, nitrite is not found in the urine. However, a subject with a suspected UTI will produce nitrite in their urine since the bacteria present will convert nitrate into nitrite. Traditionally, nitrite is monitored by urinary dipsticks that are either read by eye or using a reflectance spectrophotometer. Both methods provide a semiquantitative positive or negative result at best. In this paper, we described a novel, affordable, portable transmission-based colorimeter for the quantitative measurement of nitrite. A unique permutation of the Griess reaction was optimized for the clinical detection of nitrite in urine and is reported. By using nitrite spiked in a salt buffer, artificial, and human urine samples, the performance of the colorimeter was evaluated against dipsticks read using two commercial dipstick analyzers, Urisys 1100 (Roche Diagnostics) and Clinitek Status+ (Siemens Medical Solutions). The colorimeter was able to detect the clinically relevant range of nitrite from 0.78 to 200 µM in a salt buffer. The detection limit in artificial urine was determined as 1.6 µM, which is ∼16× more sensitive than commercial dipstick reflectance analyzers, enabling the possibility for earlier detection of urinary infections. The colorimeter is assembled using off-the-shelf components (<$80) and controlled by a smartphone application via low-energy bluetooth. It has a built-in color correction algorithm and is designed to enable for a turbidity correction in samples containing bacteria or other cellular debris as well. The mobile application can display the nitrite concentration for a single sample or display the results over a period of time. Tracking urinalysis results longitudinally can help identify trends such as increases in nitrite concentrations over an individual's baseline and identify possible infections earlier. While the detection of nitrite was showcased here, this portable analyzer can be expanded to other colorimetric-based chemistries to detect a panel of biomarkers, which can improve the overall sensitivity and specificity of the desired assay.

Nanoscale plasmonic interferometers for multispectral, high-throughput biochemical sensing.

In this work, we report the design, fabrication, and characterization of novel biochemical sensors consisting of nanoscale grooves and slits milled in a metal film to form two-arm, three-beam, planar plasmonic interferometers. By integrating thousands of plasmonic interferometers per square millimeter with a microfluidic system, we demonstrate a sensor able to detect physiological concentrations of glucose in water over a broad wavelength range (400-800 nm). A wavelength sensitivity between 370 and 630 nm/RIU (RIU, refractive index units), a relative intensity change between ~10(3) and 10(6) %/RIU, and a resolution of ~3 × 10(-7) in refractive index change were experimentally measured using typical sensing volumes as low as 20 fL. These results show that multispectral plasmonic interferometry is a promising approach for the development of high-throughput, real-time, and extremely compact biochemical sensors.

Expression of recombinant MDA-BF-1 with a kinase recognition site and a 7-histidine tag for receptor binding and purification.

Prostate cancer metastasizes predominantly to bone, where it induces osteoblastic lesions. Paracrine factors secreted by the metastatic cancer cells are thought to mediate these events. We previously isolated a novel bone metastasis-related factor (MDA-BF-1) from bone marrow aspirate samples from patients with prostate cancer and bone metastasis, and found that this factor stimulated osteoblast differentiation, possibly by interacting with a receptor on the osteoblasts. Identifying this putative MDA-BF-1 receptor biochemically requires the expression of MDA-BF-1 for receptor binding assays and for the preparation of a ligand-affinity column. We tagged MDA-BF-1 with a peptide containing a protein kinase A phosphorylation site plus a 7-histidine sequence to facilitate the labeling of MDA-BF-1 for receptor binding assay and the binding of MDA-BF-1 to an immobilized metal affinity column. The recombinant MDA-BF-1 protein (MDA-BF1-kinase-his) was expressed in Sf9 cells using a baculovirus expression system. About 0.8 mg of purified MDA-BF1-kinase-his protein was obtained from 4 x 10(8) Sf9 cells. MDA-BF1-kinase-his can be phosphorylated by PKA with a specific activity around 10(5)cpm/mug protein. Receptor binding assays using this (32)P-labeled MDA-BF-1 showed that MDA-BF-1 bound to membranes prepared from Saos-2, an osteosarcoma cell line, and C2C12, a mouse pluripotent mesenchymal precursor cell line that can be induced to become osteoblast by BMP-2. In contrast, MDA-BF-1 did not bind to membranes from PC-3 human prostate cancer cells or HEK293 human embryonic kidney cells. These observations suggest that the MDA-BF-1 receptor is expressed in cells of osteoblastic lineage. In addition to its use as a ligand for receptor binding assays, a ligand affinity column can be prepared by binding MDA-BF1-kinase-his to an IMAC for the purification of MDA-BF-1 receptor.