Determination of nitrite in saliva using microfluidic paper-based analytical devices

Abstract

Point-of-care platforms can provide fast responses, decrease the overall cost of the treatment, allow for in-home determinations with or without a trained specialist, and improve the success of the treatment. This is especially true for microfluidic paper-based analytical devices (μPAD), which can enable the development of highly efficient and versatile analytical tools with applications in a variety of biomedical fields. The objective of this work was the development of μPADs to identify and quantify levels of nitrite in saliva, which has been proposed as a potential marker of periodontitis. The devices were fabricated by wax printing and allowed the detection of nitrite by a colorimetric reaction based on a modified version of the Griess reaction. The presented modifications, along with the implementation of a paper-based platform, address many of the common drawbacks (color development, stability, etc.) associated with the Griess reaction and are supported by results related to the design, characterization, and application of the proposed devices. Under the optimized conditions, the proposed devices enable the determination of nitrite in the 10-1000 μmol L(-1) range with a limit of detection of 10 μmol L(-1) and a sensitivity of 47.5 AU [log (μmol L(-1))](-1). In order to demonstrate the potential impact of this technology in the healthcare industry, the devices were applied to the analysis of a series of real samples, covering the relevant clinical range.

Keywords: Griess reagent; Nitrite; Paper-based microfluidic analytical device; Saliva.

Figure 1

Reaction scheme of the production of the azo dye by the interaction of the Griess reagent and nitrite.

Figure 2

The redesigned μPAD for nitrite detection in saliva showing the hydrophilic main channel, branched channels, uptake zones, and testing zones surrounded by the hydrophobic wax barrier.

Figure 3

Development of the background signal (in arbitrary units) of the Griess reagent when dried and stored in air (A), when dried under nitrogen and stored in air (B) and when dried and stored under nitrogen (C). For comparison purposes, the figure also includes the signal obtained when 10 mmol L⁻¹ nitrite was added. All experiments were conducted at room temperature.

Figure 4

Effect of temperature on the development of background signal (in arbitrary units) in the zones spotted with each component of the Griess reagent (under nitrogen). Lines added to guide the eyes.

Figure 5

Analytical calibration curve exhibiting logarithmic behavior from 10 – 1000 μmol L⁻¹ nitrite. The standard solutions were prepared in artificial saliva and analyzed using the newly designed μPAD, a scanner, and the gray channel in Photoshop®. Signal was normalized against a selected blank location on the μPAD.