2.1 Materials and chemical agents
Glucose oxidase (GOx) (Merck, Germany) from aspergillus niger, horseradish peroxidase (HRP) (Merck, Germany), 3,3,5,5-tetramethylbenzidine (TMB) (BioBasic Inc, Canada), glucose (Merck, Germany), Polyvinylpyrrolidone (PVP) (Merck, Germany), Potassium Iodide (KI), Silica nanoparticle (SiNP, Fadak, Iran), methanol (Arman Sina, Iran), and Phosphate Buffered Saline (PBS, pH=7.6) (Merck, Germany) were purchased. All chemicals were used as received. Filter paper (80 g/m2) with 50% porosity and transparent pressure-sensitive adhesive tape (TPSA) were used as the substrate (hydrophilic channel) and sealing film, respectively.
2.2 Equipment and software
A CO2 laser engraving cutting machine (Perfect, China) operating at a wavelength of 10.64 μm at 14 W was utilized for cutting cellulose filter paper. Images and videos were captured by a smartphone camera (iPhone 11, USA). CorelDRAW (2018), Tracker (5.1.3), and ImageJ (1.52a) were used for the channel design, distance-based measurement, and intensity-based image processing, respectively. The images were captured in a specific room and under a particular light and finally subtracted by a defined value in ImageJ to eliminate the background effect.
2.3 μPAD design and fabrication
Hydrophilic patterns were designed in CorelDRAW 2019. The device consisted of a sample zone (2×5 mm2), primary circular zone (diameter = 5 mm), detection zone (channel, 2×25 mm2), and absorbent circular zone (diameter = 5 mm). The designed μPAD and different zones are demonstrated in Fig. 1a. The patterns were cut by a CO2 laser engraving cutting machine (λ=10.64 μm, 14 W, 14 mm/s). Reagents were added to different zones, and the device was packed with a regular TPSA to prevent leakage and evaporation the μPAD.
2.4 Enzymatic reactions of glucose colorimetric detection
The device was dipped in a PVP solution to improve the immobilization of the chemical agents. An enzymatic solution containing 120 U/ml of GOx and 30 U/ml of HRP was added to the primary circular zone. The chromogenic solution containing TMB was dissolved in methanol and added to the channel called the detection zone. The role of the final circular zone was absorbing the extra fluid. The zone of the enzymatic solution and chromogenic agents was investigated in various experiments, and adding them to the primary circular zone and channel, respectively, was selected to achieve acceptable distance-based results.
Different concentrations of 10 μl glucose solution were added to the sample zone and wicked towards the primary circular zone by capillary action. Glucose was oxidized in the presence of GOx and the products (gluconic acid and hydrogen peroxide) entered the detection zone. H2O2 reacted with TMB in the presence of HRP, and the blue oxidized TMB was produced in the detection zone. The second circular zone absorbed excessive water and by-products. The schematic of the reactions is illustrated in Fig. 1b, Fig. 1c and Fig. 1d.
2.5 Procedure for color detection
The colorimetric assay video was captured by a smartphone camera and analyzed by ImageJ and Tracker to achieve intensity-based and distance-based results, respectively. For the intensity-based results, the frame of the video was imported into ImageJ and digitized to grayscale.
The cellulose paper is white, and the color intensity decreases after the chemical reaction, so more glucose concentration results in lower color intensity. The inverse grayscale was used to directly correlate color intensity and glucose concentration (inverse grayscale = 255 - grayscale).
The video was imported in Tracker for the distance-based results. A vector determined the wetted length or colored length at different time intervals (0 to 240 s, 0 s assumed the time that sample entered the channel). The initial and terminal points of the vector were the beginning of the channel and the end of the wetted or colored area (Fig. 3b). Finally, the vector magnitude over time diagram was plotted.
The wetted-length diagram showed the possibility of the glucose sample wicking feasibility along the channel, whereas the colored-length diagram indicated the presence of glucose and its reaction with other reagents. Therefore, when the wetted length is more than the colored length, it means the glucose had been finished during the reactions, and the sample without glucose was wicked towards the end of the channel, as can be observed in Fig. 1e.
2.6 Evaluation of different parameters
Various parameters were investigated to realize the performance of the μPAD through color intensity-based and distance-based results. The Chromogenic reagent, the immobilizer, method of adding the TMB reagent, TMB concentration, and the enzymatic solution volume were investigated.
2.6.1 Type of chromogenic reagent
TMB (15 mM) and KI (1.2 M) were used as chromogenic reagents. The color intensity and distance-based results were the evaluation factors. Each test was carried out four times for statistical clarity.
2.6.2 Type of the immobilization
The disadvantage of not using an immobilizer is that the color change could flow along the channel, and consequently, the intensity-based and distance-based results could be inaccurate. Therefore, three types of immobilizations were utilized, and wetted length, colored length, and color intensity were compared with the immobilization-free experiment.
The immobilization agents were PVP solution (concentration 1%, dissolved in PBS) and SiNP (10-15 and 100 nm, dispersed in deionized water). Each test was carried out four times for statistical clarity.
2.6.3 Method of adding TMB
The amount of chromogenic solution can affect the colored-length and intensity-based results. Four methods of adding TMB were defined:
- Adding solution by a micropipette uniformly as much as the channel became wet; it is called the uniform method (UM).
- Adding the solution by a micropipette every 5 mm of the channel; it is called the non-uniform method (NUM).
- Adding solution twice by a micropipette uniformly with an interval of 5 minutes to allow the first series of TMB solution to be dried; it is called twice uniform method (2UM).
- Dipping the secondary circular zone in the TMB solution and allowing the TMB to wick into the channel; it is called the saturated method (SM) because most of the paper pores were filled and saturated with the chromogenic solution.
Each test was carried out four times for statistical clarity.
2.6.4 The concentration of TMB
Different concentrations of TMB solutions (2.5, 3, 3.75, 5mM) were used as chromogenic reagents to have an appropriate colored length for the tear glucose concentration. Each test was carried out four times for statistical clarity.
2.6.5 The volume of enzymatic solution
The enzymatic solutions were added to the primary circular zone to investigate the effect of the enzyme volume on the color intensity-based results. For this purpose, adding 3 μl, 4 μl, and dipping the circular zone in enzymatic solution (saturated method, SM) were examined to obtain adequate volume for the solution. Each test was carried out four times for statistical clarity.
2.6.6 Distance-based biosensor evaluation
According to previous experiments, the final test was carried out by adding the 3 μl of the enzymatic solution to the primary circular zone, and the channel of the device was saturated with TMB solution, as the chromogenic factor, with the concentration of 3.75 mM. Various concentrations of glucose samples in tear range (0.1, 0.2, 0.4, 0.8, and 1.2 mM) were added to the sample zone, and the colored length was measured. Each test was carried out four times for statistical clarity.