Copy Paper as a Platform for Low-cost Sensitive Glucose Sensing

1. INTRODUCTION

Paper-based devices have gained popularity owing to their advantages of low cost, ease of use, portability, disposability, and suitability in point-of-care testing. Various paper-based devices have been explored with a wide range of applications, e.g., biomolecule sensing [1], metal detection [2], surface-enhanced Raman spectroscopy (SERS) platform [3], blood typing [4], and electrochemical biosensing platform [5]. Typically for disposable sensing applications, a cellulose-based filter paper known as Whatman paper is used as a substrate and is patterned using wax printing [6, 7]. Wax acts as a hydrophobic barrier that can guide the direction of a liquid sample or provide a confined area for chemical reaction and detection [8]. The wax-printed filter paper has been one of the most popular choices for paper-based device fabrication owing to its simplicity and low cost, and useful in the detection of biomolecules, e.g., glucose [9], protein [10], prostate-specific antigen [11], aspartate aminotransferase, and alanine aminotransferase [12]. Wax printing, however, is becoming more difficult to perform because the production of the widely used wax printer model that the majority of the researchers have been using, the Xerox ColorQube 8570, has been discontinued; thus, it is becoming difficult to purchase printers that use wax or solid ink. As a result, a new material and fabrication method for developing paper-based devices are required.

As an alternative to wax-printed filter paper, copy paper has been demonstrated to be an effective platform for disposable paper-based sensors. Soga et al. demonstrated an inkjet-printed colorimetric sensor for discrimination of volatile primary amines [13], and Yamada et al. demonstrated a dip-stick type distanced-based nickel sensor using copy paper [14]. In this study, we explored the possibility of using widely available copy papers as substrates that can be used for enhancing enzymatic reaction. As an example, the enzymatic colorimetric detection of glucose was performed on three substrates: a copy paper, filter paper, and polyethylene (PET) film. Then, the pretreatment of the paper substrate for glucose detection was optimized, and a photo box was 3D-printed to demonstrate the applicability of copy papers as a point-of-care testing (POCT) platform with a mobile phone. To the best of our knowledge, this is the first study to report enhanced colorimetric detection using copy papers, and it is expected to create opportunities for using such a widely available cheap substrate for the development of sensitive biosensors.

2. EXPERIMENTAL

2.2 Investigation of substrates

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).

3. RESULTS AND DISCUSSIONS

3.1 Investigation of the detection of glucose on different substrates

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.

3.2 Fabrication and optimization of the paper-based glucose sensor

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.

3.3 Characterization of the glucose sensor performance

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.

Table 1.

Performance of paper-based colorimetric glucose sensors using GOx and HRP enzymes.

4. CONCLUSIONS

This study demonstrated the potential of the use of widely available copy papers as substrates for developing paper-based sensors. The enzymatic colorimetric detection of glucose was performed on three different substrates and exhibited enhanced detection using copy papers. Although significantly higher signal intensity was observed on the copy paper, the mechanism was uncertain owing to unknown substances that existed in the copy paper. There are several reactions involved in glucose detection including the oxidation of glucose, reduction of H₂O₂, and oxidation of KI and it is difficult to determine exactly which part of the reaction is affected by the copy paper or its additives. In a future study, we plan to identify the components of the paper additives and observe their effects on both enzyme activity and the colorimetric reaction in depth to determine what causes the enhanced signal intensity. The study results suggested that a copy paper can be a viable cost-effective option for developing paper-based sensors for POCT using a mobile phone and 3D printed photo box. Moreover, this study helps open opportunities for the application of copy papers as substrates for paper-based devices and is expected to positively contribute to studies on paper-based sensors and biosensors.

Acknowledgments

This study was supported by the Pukyong National University Development Project Research Fund, 2022, and the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (Grant Nos. 2020R1C1C1003567 and 2022R1A5A8023404).

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