Rapid Gout Detection Method and Kit

Abstract

Gout is a form of arthritis characterized by buildup of uric acid in synovial fluid, which causes severe swelling and can harm joints, tendons, and other tissues. It affects approximately 4% of the United States population, or approximately 8.3 million people nationwide and is therefore a topic of epidemiologic consideration due to its prevalence. Gout is typically diagnosed via polarized microscopy of arthroscopically-aspirated synovial fluid, which is a costly, time-consuming, labor-intensive, and technically complex procedure, warranting a simpler and less complex method for diagnosis. Here, we propose and validate a colorimetric method which is based on the ability of uric acid to reduce silver nitrate. We also assessed how the colorimetric change can be accelerated by changing the concentration of silver nitrate or adding different silver catalysts, as well as develop a matrix bed for improved handling and ease of use. When translated to the clinic, this diagnostic method for gout will have the potential to increase diagnostic efficiency and accelerate patient care at the bedside.

Keywords: biomarkers; diagnostic; gout; monosodium urate crystals; point-of-care.

Figure 1

Rapid gout detection device prototype. The device consists of a leak-proof round well of 1.5 mm diameter on a plastic slide. The base of the well will be coated with reactive-ready silver nitrate solution and incubated overnight in a dehydrator to have dried layer of silver nitrate on the base.

Figure 2

Reduction of silver nitrate solution of increasing concentrations by crystallized urate. Urate crystals were added to 200 µL solutions of increasing concentration of silver nitrate; 20% silver nitrate solution demonstrated most effective staining.

Figure 3

Reduction of silver nitrate pre-coated stains of increasing concentrations by crystallized urate. Varying concentrations of silver nitrate solution were added to wells and allowed to dry overnight, before being removed by pipet. Amorphous uric acid powder (1 mg/200 µL) in suspension was added to each of the wells; 20% silver nitrate showed most effective staining.

Figure 4

Distinguish between urate and calcium crystals. Panel 1: Urate crystals were added to stain (20% silver nitrate and 1% alizarin red) pre-coated gout detection device. Images were taken with Iphone-6 attached with Proscope Micromobile microscope (no polarizing filter was used). A: 1× magnification; B: 10× magnification. Urate crystals are stained black. Panel 2: Urate and Calcium pyrophosphate crystals were added to 20% silver nitrate—gelatin bed. Urate crystals are stained black whereas Calcium pyrophosphate crystals are unstained.

Figure 5

Nondiamine silver stain. This figure illustrates the effectiveness of nondiamine silver staining in catalyzing silver reduction by urate. Well reagents are as follows: 1 = urate; 2 = urate, formaldehyde; 3 = urate, sodium bicarbonate; 4 = formaldehyde, sodium bicarbonate; 5 = urate, formaldehyde, sodium bicarbonate; 6 = urate, formaldehyde, sodium bicarbonate.

Figure 6

Ammoniacal silver stain. This figure illustrates the effectiveness of ammoniacal silver staining in catalyzing silver reduction by urate. Well reagents are as follows: 1 = urate; 2 = urate, formaldehyde; 3 = urate, citric acid; 4 = formaldehyde, citric acid; 5 = urate, formaldehyde, citric acid; 6 = urate, formaldehyde, citric acid.

Figure 7

Comparison of 20% silver nitrate solution and 1.14 M silver nitrate solution upon addition of urate. Top photos: 20% silver nitrate; bottom photos: 1.14 M silver nitrate solution. First column of photos: 0 min elapsed; second column of photos: 5 min elapsed.

Figure 8

Matrix bed development with 6% gelatin or 1.2% agar. Left image: 6% gelatin was mixed with 20% silver nitrate and added to wells, followed by polymerization solution (0.5 mL); right image: 1.2% agar was mixed with 20% silver nitrate and added to wells, followed by polymerization solution (0.5 mL). Upon development of the agar matrix, a color was produced, and therefore gelatin matrices will be preferentially developed for colorimetric reaction.

Figure 9

Gelatin matrix testing with urate. Gelatin-silver nitrate beds were developed as previously discussed. Urate crystal suspension and dried urate crystals were individually added to the matrix beds. Both the crystals and crystal suspension induced an effective colorimetric change upon addition to the gelatin-silver nitrate matrix.