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A mixture containing 120 U/mL glucose oxidase (GOx) (G7141; Sigma–Aldrich, St. Louis, MO, USA), 5 mg/mL potassium iodide (KI) (60399; Sigma-Aldrich) and 30 U/mL horseradish peroxidase (HRP) (P8375; Sigma–Aldrich) and 113.5 mg/mL trehalose (T9531; Sigma–Aldrich) was prepared using phosphate-buffered saline (PBS). Glucose (G7021; Sigma-Aldrich) with varying concentrations was diluted in PBS to be used as a sample for investigating substrates, sensing optimization, and the characterization of paper-based sensors.

Three substrates were investigated in this study: Whatman filter paper, PET film, and copy paper. No chemical or physical treatment was performed on the copy paper and PET film, except that they were cut into an appropriate size for the experiment (Figs. 1A and C). The filter paper was punched into circular discs using a 2-mm biopsy punch and placed on a sticky surface of a lamination film (Fig. 1B). To observe the difference in the colorimetric reaction for the three substrates, a 3 μL droplet mixture containing the reagents and glucose sample was placed on the surface of the substrates and imaged using a Samsung Galaxy S22 mobile phone camera (Samsung, Republic of Korea). Samples containing 0, 2.5, and 10 mM of glucose were compared, and all the experiments were performed in triplicates.

Fig. 1. 
Schemes and of glucose detection using (A) copy paper, (B) Whatman disc, and (C) PET film. (D) A sample containing glucose exhibited a dark brown color on the A4 paper. (E) The Whatman disc and (F) PET film exhibited a less intense yellow color (image of 10-mM glucose 10 min after the reaction).

FDM 3D printer (LUGO pro, LUGOLABS, Republic of Korea) was used to print a holder in 3D that can be attached to a mobile phone. The holder allows the consistent placement of a paper-based sensor with respect to the location of the camera for consistent imaging and helps provide a dark room to minimize errors that can arise from varying ambient light.

We have dispensed 0.5, 1, 2, and 3 μL of a mixture of chemical reagents required for glucose sensing on a paper to optimize the pretreatment volume required for glucose sensing. The reaction area on the copy paper and PET film was determined by the contact area of the enzyme droplet on each substrate. The droplets resulted in a reproducible area due to the consistent hydrophobicity of the substrates. For reagent drying, trehalose was added as a stabilizer to help retain the enzyme activity. Temperature was 17℃ and humidity was 11% and the air pressure was 1 atm (101 kPa). On the top part of the pretreated sensing zones, 0.5, 1, 2, and 3 μL of a sample containing 2.5 mM glucose were dropped using a micropipette to observe the effect of the sample volume on signal intensity. The experiments were performed in triplicate.

The images were analyzed using ImageJ (NIH ImageJ; NIH, Bethesda, MD). The average intensity of the detection zone was measured and subtracted by the average intensity of a nearby substrate to normalize the signal intensity.

To investigate the effect of different substrates on colorimetric reaction, samples containing different concentrations of glucose (0, 2.5, and 10 mM) were loaded on a copy paper, filter paper, and PET film (Fig. 1). The signal intensity of the sample containing 0 mM glucose exhibited little to almost no difference while using the filter paper and PET film; however, the signal intensity increased from 0 to 17 A.U. for the copy paper after 10 min of reaction (Fig. 2A). For samples containing 2.5 and 10 mM glucose, the intensity of the colorimetric reaction in the copy paper was significantly greater than those in the filter paper and PET film (Figs. 2B and C). Moreover, the rate of change in the signal intensity was much faster in the copy paper. We examined the data points in Fig 2 to determine how long it takes for the detection signal to reach signal-to-noise ratio (SNR) of 10, which is the point at which the detection signal intensity surpasses 10 times the standard deviation of blank. This threshold is considered sufficient for obtaining reliable measurements. When detecting 2.5 mM of glucose, it took approximately 180 s and over 10 min on filter paper and PET film respectively to reach SNR of 10. Additionally, approximately 25 s and 300 s were required for filter paper and PET film respectively when detecting 10 mM of glucose. On the other hand, less than 5 seconds were sufficient to reach the SNR of 10 when using copy paper as a substrate for the two glucose concentrations. This indicates that glucose detection on copy paper not only results in increased signal intensity but also requires a shorter time for measurements. Although the mechanism behind the enhanced colorimetric reaction is unknown, this result implies that a copy paper can be used as a substrate for a colorimetric enzyme reaction and point-of-care testing (POCT) biosensing platform. Thus, we decided to demonstrate a simple paper-based glucose sensor using a mobile phone.

Fig. 2. 
Signal intensity of glucose detection performed on different substrates. The graph shows the signal intensity with respect to time for samples containing (A) 0 mM glucose, (B) 2.5 mM glucose, and (C) 10 mM glucose.

To use paper-based sensors for POCT purposes, imaging should be consistently performed and should not be affected by ambient light. To provide an environment for repeatable measurements that can be performed using a mobile phone, a 3D-printed photo box was fabricated (Fig. 3). The box can be slid into the phone for an easy attachment; it can help attain the repeatable positioning of the paper-based sensor with respect to the camera and provide a darkroom for consistent imaging.

Fig. 3. 
3D-printed photo box attached to a mobile phone for imaging the paper sensor. The paper sensor was inserted through the slit located on the side of the 3D-printed photo box. The paper sensor was imaged in the darkroom of the 3D-printed photo box.

Preferably, reagents involved in the colorimetric reaction (i.e., glucose oxidase, HRP, and potassium iodide) should be pretreated on the paper, so that users only load the sample to detect glucose. To achieve this, different volumes of the reagents (0.5, 1, 2, 3 μL) were loaded on the copy paper and dried. Then, 0.5, 1, 2, and 3 μL of the sample containing 2.5 mM glucose were loaded to investigate the optimal pair of the pretreatment volume and sample volumes. As shown in Fig. 4, as the volumes of the pre-dried enzyme and sample increased, the intensity of the colorimetric reaction increased. However, there was no significant difference between 2 and 3 μL of the pre-dried samples. Considering the time required to dry the enzyme and sample, 2 μL of the pre-dried enzyme and 2 μL of the sample were selected for further experiments.

Fig. 4. 
Optimization results of the pre-dried enzyme and glucose sample on the copy paper.

Considering the optimized pretreatment condition and sample volume, the detection performance of the paper-based sensor was investigated. Fig. 5A shows a logarithmic relationship between the detection intensity and glucose concentration. The colorimetric intensity on the copy paper is much higher than that on the PET film, which implies that a copy paper is a promising suitable substrate for paper-based sensor development.

Fig. 5. 
(A) Intensity of glucose detection on the copy paper and PET film with respect to the glucose concentration. (B) Images of the glucose detection results performed on copy paper and PET film. (C) Intensity of glucose detection on the copy paper with respect to the glucose concentration considering a logarithmic scale for linear fitting.

To determine the limit of detection (LOD) and limit of quantification (LOQ) of the paper-based glucose sensor, detection results were plotted with respect to the glucose concentration in a logarithmic scale (Fig. 5B). The linear fit equation was obtained with an R² value of 0.989. The LOD (3 × SD/slope) and LOQ (10 × SD/slope) were 0.143 and 0.477 mM, where SD denotes the root mean square deviation. Compared to the LOD and LOQ of detection performed on PET film, which are 0.279 and 0.929 mM (R² = 0.913), this indicates that the copy paper provides more sensitive detection.

The detection performances of the recently developed paper-based glucose sensors that use GOx and HRP are listed in Table 1. The LOD achieved in this study is 0.143 mM, which is on the lower spectrum compared to previous studies. LOD and LOQ are expected to be improved with further optimization of experimental conditions, e.g., enzyme concentration and drying conditions.