Paulo T. Garcia

A handheld stamping process to fabricate microfluidic paper-based analytical devices with chemically modified surface for clinical assays

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.

Colorimetric determination of nitrite in clinical, food and environmental samples using microfluidic devices stamped in paper platforms

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%.

Enhanced Analytical Performance of Paper Microfluidic Devices by Using Fe3O4 Nanoparticles, MWCNT, and Graphene Oxide.

Spheres, tubes, and planar-shaped nanomaterials as Fe3O4 nanoparticles (MNPs), multiwalled carbon nanotubes (MWCNT), and graphene oxide (GO) were used for the first time to treat microfluidic paper-based analytical devices (μPADs) and create a biocompatible layer with high catalytic surface. Once glucose measurements are critical for diabetes or glycosuria detection and monitoring, the analytical performance of the proposed devices was studied by using bienzymatic colorimetric detection of this carbohydrate. The limit of detection values achieved for glucose with μPADs treated with MNPs, MWCNT, and GO were 43, 62, and 18 μM, respectively. The paper surface modification solves problems associated with the lack of homogeneity on color measurements that compromise the sensitivity and detectability levels in clinical diagnosis.

Polyester‐toner electrophoresis microchips with improved analytical performance and extended lifetime

This paper reports the fabrication of polyester‐toner (PT) electrophoresis microchips with improved analytical performance and extended lifetime. This has been achieved with a better understanding about the EOF generation and the influence of some parameters including the channel dimensions (width and depth), the injection mode, and the addition of organic solvent to the running buffer. The analytical performance of the PT devices was investigated using a capacitively coupled contactless conductivity detector and inorganic cations as model analytes. The proposed devices have exhibited EOF values of (3.4 ± 0.2) × 10−4 cm2 V−1 s−1 with good stability over 25 consecutive runs. It has been found that the EOF magnitude depends on the channel dimension, i.e. the wider the channel, the higher the EOF value. The separation efficiency for inorganic cations ranged from 13 000 to 50 000 plates/m. The LOD found for K+, Na+, and Li+ were 4.2, 7.3, and 23 μM, respectively. In addition, the same PT device has been used by three consecutive days. Lately, due to improved analytical performance, it was carried out by the first time the detection of inorganic cations in real samples such as energetic drinks and pharmaceutical formulations.

Paper-Based Colorimetric Biosensor for Tear Glucose Measurements

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.

Avaliação de dispositivos de captura de imagens digitais para detecção colorimétrica em microzonas impressas

This report describes a study about the feasibility of using a conventional digital camera, a cell-phone camera, an optical microscope, and a scanner as digital image capture devices on printed microzones. An array containing nine circular zones was drawn using graphics software and printed onto transparency film by a laser printer. Due to its superior analytical performance, the scanner was chosen for the quantitative determination of Fe2+ in pharmaceutical samples. The data achieved using scanned images did not differ statistically from those attained by the reference spectrophotometric method at the confidence level of 0.05.

Versatile fabrication of paper-based microfluidic devices with high chemical resistance using scholar glue and magnetic masks

Simple methods have been developed for fabricating microfluidic paper-based analytical devices (μPADs) but few of these devices can be used with organic solvents and/or aqueous solutions containing surfactants. This study describes a simple fabrication strategy for μPADs that uses readily available scholar glue to create the hydrophobic flow barriers that are resistant to surfactants and organic solvents. Microfluidic structures were defined by magnetic masks designed with either neodymium magnets or magnetic sheets to define the patter, and structures were created by spraying an aqueous solution of glue on the paper surface. The glue-coated paper was then exposed to UV/Vis light for cross-linking to maximize chemical resistance. Examples of microzone arrays and microfluidic devices are demonstrated. μPADs fabricated with scholar glue retained their barriers when used with surfactants, organic solvents, and strong/weak acids and bases unlike common wax-printed barriers. Paper microzones and microfluidic devices were successfully used for colorimetric assays of clinically relevant analytes commonly detected in urinalysis to demonstrate the low background of the barrier material and generally applicability to sensing. The proposed fabrication method is attractive for both its ability to be used with diverse chemistries and the low cost and simplicity of the materials and process.

Batch injection analysis towards auxiliary diagnosis of periodontal diseases based on indirect amperometric detection of salivary α-amylase on a cupric oxide electrode

This study describes, for the first time, the use of a batch injection analysis system with amperometric detection (BIA-AD) to indirectly determine salivary α-amylase (sAA) levels in saliva samples for chronic periodontitis diagnosis. A chemical/thermal treatment was explored to generate a CuO film on a Cu electrode surface. This procedure offered good stability (RSD = 0.3%), good repeatability (RSD < 1.3%) and excellent reproducibility (RSD < 1.5%). The sAA concentration levels were determined based on the detection of maltose produced by enzymatic hydrolysis of starch. The analytical performance was investigated, and a linear correlation was observed for a maltose concentration range between 0.5 and 6.0 mmol L-1 with a correlation coefficient equal to 0.999. The analytical sensitivity and the limit of detection were 48.8 μA/(mmol L-1) and 0.05 mmol L-1, respectively. In addition, the proposed system provided an excellent analytical frequency (120 analysis h-1). The clinical feasibility of the proposed method was investigated by the determination of sAA levels in four saliva samples (two from healthy control persons (C1 and C2) and two from patients with chronic periodontitis (P1 and P2)). The accuracy provided by the BIA-AD system ranged from 93 to 98%. The sAA concentration levels achieved for each sample were compared to the values found by spectrophotometry and there was no statistically significant difference between them at a confidence level of 95%. Finally, the method reported herein emerges as a simple, low cost and promising tool for assisting periodontal diseases diagnosis.

Enhanced Performance of Colorimetric Biosensing on Paper Microfluidic Platforms Through Chemical Modification and Incorporation of Nanoparticles

This chapter describes two different methodologies used to improve the analytical performance of colorimetric paper-based biosensors. Microfluidic paper-based analytical devices (μPADs) have been produced by a stamping process and CO2 laser ablation and modified, respectively, through an oxidation step and incorporation of silica nanoparticles on the paper structure. Both methods are employed in order to overcome the largest problem associated with colorimetric detection, the heterogeneity of the color distribution in the detection zones. The modification steps are necessary to improve the interaction between the paper surface and the selected enzymes. The enhanced performance has ensured reliability for quantitative analysis of clinically relevant compounds.