This paper describes the development and use of a handheld and lightweight stamp for the production of microfluidic paper-based analytical devices (μPADs). We also chemically modified the paper surface for improved colorimetric measurements. The design of the microfluidic structure has been patterned in a stamp, machined in stainless steel. Prior to stamping, the paper surface was oxidized to promote the conversion of hydroxyl into aldehyde groups, which were then chemically activated for covalent coupling of enzymes. Then, a filter paper sheet was impregnated with paraffin and sandwiched with a native paper (n-paper) sheet, previously oxidized. The metal stamp was preheated at 150 °C and then brought in contact with the paraffined paper (p-paper) to enable the thermal transfer of the paraffin to the n-paper, thus forming the hydrophobic barriers under the application of a pressure of ca. 0.1 MPa for 2 s. The channel and barrier widths measured in 50 independent μPADs exhibited values of 2.6 ± 0.1 and 1.4 ± 0.1 mm, respectively. The chemical modification for covalent coupling of enzymes on the paper surface also led to improvements in the colour uniformity generated inside the sensing area, a known bottleneck in this technology. The relative standard deviation (RSD) values for glucose and uric acid (UA) assays decreased from 40 to 10% and from 20 to 8%, respectively. Bioassays related to the detection of glucose, UA, bovine serum albumin (BSA), and nitrite were successfully performed in concentration ranges useful for clinical assays. The semi-quantitative analysis of all four analytes in artificial urine samples revealed an error smaller than 4%. The disposability of μPADs, the low instrumental requirements of the stamp-based fabrication, and the improved colour uniformity enable the use of the proposed devices for the point-of-care diagnostics or in limited resources settlements.

This paper describes the modification of microfluidic paper-based analytical devices (μPADs) with chitosan to improve the analytical performance of colorimetric measurements associated with enzymatic bioassays.

This study reports the use of microfluidic paper-based analytical devices (μPADs) associated with colorimetric detection for the determination of nitrite in clinical, food and environmental samples. μPADs were fabricated by a simple and fast stamping process in a geometry containing eight circular detection zones and one central zone to sample inlet interconnected by microfluidic channels. The colorimetric determination of nitrite was performed through the modified Griess reaction. Detection zones were spotted with a 0.75 μL aliquot of a solution containing 50 mM sulfanilamide, 1.2 M hydrochloric acid and 4 mM N-(1-naphthyl)ethylenediamine. The monitoring of the background colorimetric response revealed good stability over 12 h for devices stored in the absence of light. After the addition of standard or real samples, the resulting images were captured with a scanner, converted to a color scale and analyzed in the magenta channel. The analytical sensitivity and the limit of detection achieved after a preconcentration stage were 0.56 (AU μM⁻¹) and 5.6 μM, respectively. The preconcentration provided an enrichment factor of ca. 3.2 times. The concentration levels of nitrite were successfully determined in saliva, preservative water, ham, sausage and river water samples. The concentration levels attained for each sample using μPADs were compared to the values found by spectrophotometry and there was no significant difference from one another at a confidence level of 95%.

This paper describes a paper-based colorimetric biosensor for measuring glucose concentration levels in human tear samples. Colorimetric biosensors were wax printed on paper platforms and modified with chitosan previously prepared in acetic acid. The proposed device was explored to measure the glucose levels in human tear samples using 3,3′,5,5′-tetramethylbenzydine (TMB) as the chromogenic reagent. The paper-based colorimetric biosensor exhibited a linear behavior for the glucose concentration range between 0.1 and 1.0 mM. The achieved analytical sensitivity and limit of detection (LOD) were 84 AU/mM and 50 µM, respectively. Moreover, the device provided analytical reliability and no statistical difference when compared to the data recorded with a commercial glucometer. The proof-of-concept of our device was successfully demonstrated by measuring the glucose levels in six tear samples from nondiabetic subjects. In general, the results showed that the colorimetric biosensor has noticeable potential to be used as a powerful tool for tear glucose monitoring, since this fluid offers lower potential interferences, non-invasive sample collection and is pain-free. Furthermore, the proposed device could facilitate the treatment of diabetic patients who need constant control of glucose levels and cannot tolerate multiple finger sticks per day.