Simultaneous quantification of multiple biomarkers on a self-calibrating microfluidic paper-based analytic device

Journal Pre-proof Simultaneous quantification of multiple biomarkers on a self-calibrating microfluidic paper-based analytic device SeJin Kim, Dami Kim, Sanghyo Kim PII:

S0003-2670(19)31308-X

DOI:

https://doi.org/10.1016/j.aca.2019.10.068

Reference:

ACA 237202

To appear in:

Analytica Chimica Acta

Received Date: 29 May 2019 Revised Date:

30 September 2019

Accepted Date: 24 October 2019

Please cite this article as: S. Kim, D. Kim, S. Kim, Simultaneous quantification of multiple biomarkers on a self-calibrating microfluidic paper-based analytic device, Analytica Chimica Acta, https:// doi.org/10.1016/j.aca.2019.10.068. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.

Simultaneous quantification of multiple biomarkers on a self-calibrating microfluidic paper-based analytic device

SeJin Kim, Dami Kim, and Sanghyo Kim*

Department of Bionanotechnology, Gachon University, Seongnam, 461-701, Republic of Korea.

*Corresponding Author: Prof. Sanghyo Kim, E-mail: [email protected], Tel: +82-31-750-8554; Fax: +82-31-750-8819 1

ABSTRACT In this study, we developed a point-of-care assay platform with simultaneous detection and self-calibration capabilities for multiple targets based on a microfluidic paper-based analytical device (µPAD). This system is easily manufactured using a wax printing method on chromatographic paper. The design pattern consists of a zone of detection and a calibrant zone for controlled loading using wax barriers with different thicknesses. We showed the utility and applicability of this approach by a proof-of-concept study for two clinically important markers: glucose and lactate. With the naked eye, the results could be fully distinguished and recorded to evaluate the analytical performance with a flatbed scanner. The detection limits of glucose and lactate were 0.3125 mM and 0.2975 mM, respectively, and simultaneous detection was possible from a small sample (0.4 µL) with high sensitivity. Furthermore, this device has a self-calibration function, which minimizes the influence of environmental conditions (i.e., ambient light intensity, temperature, humidity, and pressure). Therefore, the developed multiplex paper-based device is promising for clinical multianalyte point-of-care testing since it is easy to manufacture, cost-effective, user-friendly, and highly sensitive. Keywords: Colorimetric; Glucose; Paper-based devices; Point-of-care, Self-calibration;

2

1. Introduction Microfluidic paper-based analytical devices (µPADs) have attractive features, such as low cost, biodegradability, biocompatibility, flexibility, accessibility, and ease of production [1-4]. These instruments are therefore, commonly used in different fields, such as food safety, environmental monitoring, biochemical analysis, and clinical diagnosis [5-9]. Paper-based methods have many advantages: they are simple, fast, cost-effective, easy, and profitable in remote communities and developing countries and have high-throughput and lowconsumption of the sample. They also provide high contrast for colorimetric assays, which makes the measurement of target analytes highly sensitive [10]. However, conventional colorimetric methods for providing qualitative results are greatly affected by environmental conditions such as ambient light intensity, temperature, humidity, and pressure [11-14]. Therefore, an external calibration curve is essential to minimize errors and to provide highquality quantitative results. Generally, self-calibrated devices have pre-stored standard solutions of known concentrations of the target analyte to be analyzed [11, 12, 14]. Selfcalibrated devices, therefore, must be able to simultaneously detect multiple standard analytes and unknown samples in the same environment and in parallel. Unfortunately, to date, few studies have reported user-friendly, simultaneous detection on a single device. Therefore, it is necessary to develop a convenient, sensitive, and inexpensive platform that can accurately detect multiple biomarkers. Here, we demonstrate a device that can simultaneously measure glucose and lactate using a simple wax-printing patterning technique. Volume-sensitive barriers were formed using different wax patterning thicknesses to allow controlled loading of the detection reagent and calibrant solution. The developed device has a series of standard analyte solutions pre-stored 3

in the paper chip during manufacturing, so users can easily obtain the calibration curve and can quantify multiple markers more accurately. Furthermore, the developed µPADs are based on the detection of H2O2 produced by an analyte-specific oxidase. Therefore, several biomarkers such as glucose, lactate, uric acid, cholesterol, ethanol, and cortisol can be analyzed in these qualitative and quantitative analytical devices [15-17] (Scheme 1).

2. Experimental 2.1. Chemicals and materials D-(+)-glucose, sodium L-lactate, glucose oxidase (GOx) (100 U/mg), lactate oxidase (LOx) (40 U/mg), horseradish peroxidase (HRP) (250 U/mg), 4-aminoantipyrine (4-AAP), sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS), D-(+)-trehalose dihydrate (≥ 98.5%), sodium chloride, magnesium chloride, calcium chloride, D-(−)-fructose, D-lactose monohydrate, sucrose, uric acid, L-cysteine, phosphate buffered saline powder (10 mM, pH 7.4) and human blood serum (H4522) were purchased from Sigma-Aldrich. Bovine serum albumin (BSA) was purchased from MP Biomedicals. Whatman Grade 1 Chr Cellulose Chromatography Paper (5 cm × 100 m, thickness = 0.18 mm) was purchased from Thermo Fisher Scientific. All reagents were used as received and prepared in ultrapure water (18 mΩ⋅cm, Millipore) without further purification.

2.2. Fabrication and preparation of the µPADs The µPAD was produced by a wax patterning manufacturing process. The hydrophobic 4

pattern template was prepared using AutoCAD 2016 and printed on a chromatographic filter paper using a ColorQube 8570 wax printer. Then, the wax-printed paper was heat-treated at 120 °C for 90 seconds and cooled at 25 °C. As a result, the wax melted and penetrated through the paper, forming a hydrophobic barrier inside the paper (Fig. 1a). In addition, thin hydrophobic barriers were designed for controlled loading of reagents and samples (Fig. 1a). The channel length and width of the µPAD are 4 mm and 3 mm, respectively. In addition, 10 circular detection zones were manufactured with a diameter of 4 mm. A transparent adhesive film was machined to 26 × 26 mm using a CO2 laser marking machine (DONGIL, MC30). The upper transparent adhesive film has a buffer inlet and a sample inlet with diameters of 3 mm and 2 mm, respectively. Finally, µPADs were obtained by laminating transparent adhesive films (top and bottom layer) and the wax-patterned paper (middle layer) (Fig. 1a).

2.3. Preparation of calibrant-loaded µPADs Glucose was detected using a mixture of GOx (100 U/mL) and HRP (100 U/mL) prepared in 0.3 M trehalose in PBS (pH = 7.4). Lactate was detected using LOx (20 U/mL) instead of GOx. Solutions containing 4-AAP (20 mM) and DHBS (20 mM) were dissolved in water. All solutions were stored at 4 °C until use. First, each detection zone was loaded with 1% BSA in PBS (pH = 7.4) and completely dried. Enzyme and chromogen (assay reagent) were freshly mixed in a 1:1:1:1 ratio, after which 0.5 µL of assay reagent was prespotted on each detection zone. Calibrant (glucose and lactate) solutions (1.25−5 mM) were prepared from a stock solution of 40 mM in water. Afterwards, 0.4 µL of a series of calibrant solutions was embedded in the calibrant zone and completely dried for 10 min in the dark (Fig. 1b). 5

2.4. Assay procedures for glucose and lactate Fig. 1c shows the experimental procedure for the simultaneous detection of glucose and lactate in an unknown sample using the calibrant-loaded µPAD. First, 0.4 µL of the sample was injected into the sample inlet; then, 30 µL of PBS (pH 7.4) was injected into the buffer inlet. Thereafter, the reaction proceeded at room temperature for 10 minutes, and the data was analyzed.

2.5. Data (Image) analysis The colorimetric signals obtained in the detection zones were recorded using a flatbed scanner (Canon LIDE-120), and the scan was performed in color mode at a resolution of 600 dpi (dots-per-inch) and saved in JPEG format. Images were cropped to the same size (625 × 625 pixels) to obtain the same number of pixels for data analysis. The color information was obtained with an image analysis software program (ImageJ ver. 1.52). With ImageJ, a circle covering the majority of the uniform color within each reaction zone was used to determine each value. Then, the plugin titled RGB Measure was used to determine the mean R, G, and B values for the pixels contained within the circle (Fig. S1). Color intensity was calculated using Eq 1. Color intensity =

(

−

) +(

−

) +(

6

−

) (1)

3. Results and discussion 3.1. Optimization of wax barrier and reagent volumes Our µPADs have two barriers: the hydrophobic main barrier and hydrophobic thin barriers. The thicknesses of the hydrophobic main barrier and hydrophobic thin barriers were selected to be 0.20 mm and 0.05 mm, respectively (Fig. S2). The 0.20 mm thick barrier served well as a barrier even with excess solution volume. On the other hand, the 0.05 mm thick barrier lost its function above a certain solution volume. Therefore, thin barriers formed barriers for a small volume of assay reagent (0.5 µL) and calibrant solution (0.4 µL), enabling controlled loading. Meanwhile, it served as a passive channel for a large volume of buffer solution. To demonstrate the effect of the controlled deposition and passive channel, we visualized loading using red and blue inks (Fig. S3). In addition, the optimal volume of the inlet buffer was selected to be 30 µL (Fig. S3). Compared to uPAD with no barrier, uPAD with barrier showed more controlled loading capacity.

3.2. Calibration curve of glucose and lactate First, we used modified µPADs to observe the color intensity over a range of concentrations (0.625−40 mM) (Fig. 2). These modified devices had a similar design to that of the proposed µPADs, with the exception of sample inlets and modifications of the thin barriers. Here, detection zones 5 and 6 were used as blanks. In the calibrant zone, each calibrant (glucose or lactate) was pre-stored according to the purpose of the detection chip. We first observed the color conversion effect of applying BSA on the detection zone. Glucose

7

detection results did not significantly differ upon the addition of BSA (Fig. 2a, b). In contrast, the lactate assay showed significant differences in BSA treatment (Fig. 2c, d). This is consistent with results of previous studies that the activity of lactate oxidase decreases dramatically when it is dried [18]. It is believed that the neutral protein, BSA, helps in increasing the thermostability of the lactate oxidase. Under optimized conditions, color intensity and concentration in the glucose assay showed linearity ranging from 0.3125−5.0 mM and the LOD was 0.2975 mM. For the lactate assay, the concentration was linear from 0.3125−5.0 mM and the LOD was 0.2656 mM. The LOD is given as 3S0/S, where S0 is the standard deviation of the blank measurement (n = 8) and S is the slope of the calibration curve. Here, we present an absolute quantity that can be detected by multiplying the LOD and sample volume for objective performance comparisons in existing paper devices (Table 1). The developed µPADs have sufficient detection capability, even at very small sample volumes. In addition, we extracted information on repeatability and reproducibility by comparing zone to zone and chip to chip. In terms of repeatability, the relative standard deviation (RSD) found for simultaneous analysis (n = 4) on a single chip was 4.93% and 4.51% in glucose and lactate, respectively. In contrast, the RSD values of the chip to chip (n = 8) were 6.86% and 5.65% in glucose and lactate, respectively. These results suggest that the proposed self-calibration µPADs provide excellent repeatability and reproducibility.

3.3. Simultaneous detection of glucose and lactate We prepared a solution containing 5.0 mM of glucose and lactate to show the simultaneous detection of glucose and lactate in the proposed µPADs. The solution mixture 8

was injected into the sample inlet (0.4 µL), and 30 µL of PBS (pH 7.4) was injected into the buffer inlet, followed by a 10-min reaction at room temperature. During this time, the 0.4 µL solution gets divided into two channels and has a 2-fold dilution effect relative to the calibrant. Fig. 3a shows the scanned image after 10 minutes. Fig. 3b and c show the calibration curves of glucose and lactate, respectively, and the concentrations in the mixed samples. The solutions prepared at known concentrations showed values within the error range of the linear regression curve and confirmed the possibility of simultaneous detection of glucose and lactate.

3.4. Simultaneous detection of glucose and lactate in serum samples We conducted an experiment to verify the operability of the proposed µPADs in real human serum samples. Glucose concentrations in healthy human serum samples range from 3.6−5.8 mM and lactate concentrations range from 1−20 mM [19, 20]. According to the manufacturer’s instruction (Sigma Aldrich), human serum without preservatives and additives extracted from healthy persons was collected. Therefore, we can assume that the purchased serum sample contained a normal physiological concentration of glucose. Human serum samples (0.4 µL) were injected into the sample inlet, and 30 µL PBS (pH 7.4) was injected into the buffer inlet. The results were analyzed after 10 min reaction at room temperature. Fig. 3d shows the scanned image after 10 minutes. Fig. 3e and f show linear glucose and lactate curves, respectively, at reasonable concentrations in human serum samples. Each concentration was calculated by taking the linear regression curve and the dilution factor into account. The glucose and lactate concentrations in the serum samples were 5.78 ± 0.58 mM 9

and 1.68 ± 0.30 mM, respectively. Both glucose and lactate concentrations were in the normal range and were in good agreement with those reported in the literature. Furthermore, the glucose concentration using a commercialized blood glucose meter (Roche ACCU-CHEK) was measured to be 5.51 ± 0.20 mM. The accuracy of the developed method was further evaluated by spike and recovery experiments. For this purpose, two known amounts of glucose (1 and 2 mM) and lactate (2 and 4 mM) were added to serum samples, respectively. The recoveries calculated from the spiked concentrations in serum samples were 95.64 − 103.96 % and 88.44 − 105.93 % for glucose and lactate, respectively. This indicates that the developed µPADs are accurate and can be effectively applied to monitor glucose and lactate levels in real human serum samples.

3.5. Selectivity and stability of the μPADs The selectivity of the self-calibrated µPADs was evaluated using other potential interfering substances (fructose, lactose, sucrose, NaCl, MgCl2, CaCl2, L-cysteine, and uric acid) as control samples. No significant color conversion was observed in the analysis using the control samples (Fig. 4a). These results suggest that the enzyme catalysis is highly selective and can be used to analyze human serum samples directly. The storage stability of the developed µPAD was further investigated in consecutive 30 days (Fig 4b). The prepared µPADs were stored at 4°C in dark, dry, sealed condition. No significant change in the colorimetric responses was observed. The results confirmed that the device can be stably maintained at 4°C for at least one month. Thus, the storage stability of the µPAD is acceptable and the device is available for clinical diagnostics and point-of-care testing 10

(POCT).

4. Conclusion In conclusion, we demonstrated a simple approach for simultaneous quantification of glucose and lactate in serum samples with µPADs that have self-calibration capabilities and are produced by wax printing. The proposed self-calibration µPADs can be a realistic platform to meet the demands of portability, quantitative capabilities, low cost, and user-friendliness in the field of medical health and environmental monitoring. Thus, this paper-based system not only allows semi-quantitative result analysis with the naked eye but can also provide quantitative results analysis using smartphone cameras in the future, providing a potential field diagnosis platform for disease monitoring in remote communities and developing countries.

Acknowledgements This research was supported by the R&D Program for Society of the National Research Foundation (NRF) funded by the Ministry of Science, ICT of Korea (2015M3A9E2031372).

Competing financial interests The authors declare no competing financial interests.

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Appendix A. Supplementary material

12

Fig. S1 to Fig. S3 are available in Supplementary information.

13

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Figure Captions Scheme. 1. Bienzymatic colorimetric assay diagram for detecting glucose and lactate.

Fig. 1. Fabrication and preparation of the µPADs. (a) A perspective view of the device before assembly. The device comprises two inexpensive, lightweight, and readily available components: transparent adhesive film (top and bottom layer) and Whatman grade 1 Chromatography paper (middle layer). (b) Embedding strategy of assay reagents and calibrants. Glucose detection part—detection zone (1−5): GOx, HRP, 4-AAP, and DHBS; calibrant zone (1’−4’): glucose calibrant; and calibrant zone (5’): sample. Lactate detection part—detection zone (6−10): LOx, HRP, 4-AAP, and DHBS; calibrant zone (7’−10’): lactate calibrant; and calibrant zone (6’): sample. (c) Schematic illustration of experimental assay procedures for simultaneous detection of glucose and lactate. Fig. 2. Analytical performance of µPADs without and with BSA. The inset shows the scanned image and the linear calibration curve of glucose at the range of 0.3125 to 5.0 mM. Analytical curves for the glucose assay without BSA (a) and with BSA (b). Analytical curves for the lactate assay without BSA (c) and with BSA (d). All values represent the mean and standard deviation of four independent experiments. Fig. 3. Simultaneous detection of glucose and lactate. (a) A scanned image of a known concentration solution (5 mM glucose and 5 mM lactate) experiment. (b) Glucose concentration with linear interpolation and dilution factor was 4.88 ± 0.88 mM. (c) The lactate concentration was 5.90 ± 0.92 mM based on linear interpolation and dilution factor. (d)

19

Experimental results from applying a human serum sample. (e) The glucose concentration of the plasma samples with linear interpolation and dilution factor was 5.78 ± 0.58 mM. (f) The lactate concentration of plasma samples with linear interpolation and dilution factor was 1.68 ± 0.30 mM. All values represent the mean and standard deviation of four independent experiments. Fig. 4. Specificity and stability test. (a) Specificity for various analytes (fructose, lactose, sucrose, NaCl, MgCl2, CaCl2, L-cysteine, and uric acid). All analytes were tested at a concentration of 10 mM. (b) Stability test for fabricated µPADs stored at 4°C in dark conditions for 1 month. Glucose and lactate were tested at a concentration of 5 mM. All values represent the mean and standard deviation of four independent experiments.

20

Graphical Abstracts

21

Scheme. 1.

22

Fig. 1.

23

Fig. 2.

24

Fig. 3.

25

Fig. 4.

26

Table 1. Comparison of LODs and sample volume for colorimetric glucose and lactate assays performed on paper devices. Sample Reader

LOD

Analyte (mM)

Range

Calibration

Multiplexing Channel

(nmol) (µL)

Glucose Scanner 0.2975

Self-

Quantity* Volume

Device

Linear

0.4

(mM) 0.119

****

O

O

**

0.3125−2.500

This O

Lactate

Ref. ***

work

Scanner 0.2656

0.4

0.106

0.3125−2.500

Glucose Camera 0.5000

10.0

5.000

0−5

X

O

O

[21]

Glucose Scanner 2.8000

5.0

14.000

3−50

X

X

O

[22]

Glucose Scanner 0.5000

20.0

10.000

0.5−100

X

X

X

[23]

Glucose Camera 0.2130

15.0

3.195

0.3−1.0

X

O

O

[24]

Glucose Camera 0.1500

20.0

3.000

0−20

X

X

O

[25]

Glucose Scanner 0.5000

10.0

5.000

0.5−10

Lactate

[26] X

O

O

0.6300

10.0

6.300

0.63−3.75

Glucose Camera 1.3870

10.0

13.870

0−27.75

X

O

O

[27]

Glucose Camera 0.7000

20.0

14.000

0−10

X

X

O

[28]

0.9000

3.0

2.700

3.0−30

X

X

X

[29]

Glucose Camera 0.3000

2.0

0.600

1.0−11

O

X

O

[14]

Glucose Scanner 0.5000

1.0

0.500

0.5−3.0

X

O

O

[30]

Lactate

1.0

0.500

0.5−3.0

X

O

O

InGlucose house

0.5000

27

Glucose Scanner 0.0230

70.0

1.610

0.1−1.0

X

O

O

[31]

Glucose Scanner 0.4000

6.0

2.400

0.5−5.0

X

X

O

[32]

Lactate

Camera 0.1000

50.0

5.000

0.6−10

X

X

X

[33]

Glucose Scanner 0.0500

5.0

0.250

0.1−1.0

X

X

O

[34]

Glucose Scanner 0.0205

2.0

0.041

0.056−1.249

X

X

X

[35]

Glucose Camera 0.0200

5.0

0.100

0−1

X

X

O

[36]

Glucose Camera 0.0140

3.0

0.042

0.02−4.0

X

X

X

[17]

Glucose Camera 0.0100

10.0

0.100

0.05−20

X

X

O

[37]

Glucose Camera 0.0025

10.0

0.025

0.0106−0.1500

X

X

O

[38]

Glucose Camera 0.0600

40.0

2.400

0.1−2.0

X

O

O

[10]

4.0

0.083

0.1388−2.7754

X

X

O

[15]

InGlucose

0.0207 house

Abbreviations: LOD, limits of detection; * Product of LOD and sample volume. Represents an absolute quantity that can be detected. ** Availability of calibration curve on a single chip. *** Availability of multiple target analysis on a single chip. **** Isolation of reaction zone and sampling zone.

28

Highlights •

Point-of-care assay platform with simultaneous detection and self-calibration capabilities was developed.

•

As proof of concept, enzymatic colorimetric glucose and lactate detection were successfully performed.

•

This paper-based biosensor provides low detection limit, good accuracy, and high specificity for the detection of glucose and lactate in human serum.

•

This approach can be a promising tool for disease screening in medical diagnostics.

Declaration of interests ☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: