Development of a microdevice for facile analysis of theophylline in whole blood by a cloned enzyme donor immunoassay

Keine Nishiyama a, Kanako Sugiura b, Noritada Kaji cdf, Manabu Tokeshi *ef and Yoshinobu Baba bfg
aGraduate School of Chemical Sciences and Engineering, Hokkaido University, Kita 13 Nishi 8, Kita-ku, Sapporo 060-8628, Japan
bDepartment of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
cDepartment of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
dJapan Science and Technology Agency, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
eDivision of Applied Chemistry, Faculty of Engineering, Hokkaido University, Kita 13 Nishi 8, Kita-ku, Sapporo 060-8628, Japan. E-mail: tokeshi@eng.hokudai.ac.jp; Fax: +81 11 706 6745; Tel: +81 11 706 6744
fImPACT Research Center for Advanced Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
gHealth Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu 761-0395, Japan

Received 17th October 2018 , Accepted 10th December 2018

First published on 11th December 2018


We have developed a microdevice for therapeutic drug monitoring. In this device, dispensing of sample and reagent was accomplished by simple manual operation of a syringe. Moreover, for a simple and rapid measurement, we used cloned enzyme donor immunoassay as a detection principle. These features and the reagent that is enclosed in microdevice beforehand make it possible to complete the facile analysis. In this paper, our model analyte was 1,3-dimethylxanthine (theophylline), a kind of bronchodilator. The fluorescence measurement of theophylline in whole blood was achieved with the limit of detection of 0.73 μg mL−1. This microdevice provides rapid analysis (4 min), requires only a small volume of sample (2 μL) and features simple operation; hence, it is readily applicable to point of care testing.


Introduction

In recent years, microdevices have been drawing intense research interest because of their potential applications to point of care testing (POCT).1–4 POCT is medical diagnostic testing performed at a patient's bedside, and it has attracted much attention because it contributes to efficient and rapid diagnosis. The methods used in POCT are required to have low invasiveness, high reproducibility, high accuracy, high sensitivity, high quantitative property, and high adaptability, and be rapid and easy-to-use. Microdevices are palm-sized devices and they have good potential to measure a small volume of sample in a short time by integration of analytical functions. Therefore, microdevices are suitable for achieving the above requirements.

Therapeutic drug monitoring (TDM) is a medical diagnosis field that can utilize the POCT advantages. In TDM, factors related to therapeutic effects and side effects such as blood drug concentration are monitored. Because individual treatments can be provided for each patient, such monitoring is indispensable for the proper use of drugs and risk management.5–7 TDM is conducted for drugs whose blood concentration is correlated mainly with the development of therapeutic effects and side effects; however there are few TDM systems that can measure such concentrations rapidly at a patient's bedside with a small amount of sample. Therefore, a POCT system that meets these requirements is highly desired.

In general, immunoassays are used as analytical methods in various fields such as medical diagnosis including POCT and on-site environmental analysis.8 Immunoassays are classified into homogeneous and heterogeneous types. Although heterogeneous methods generally have high sensitivity, they require physical separation of antibody–antigen complexes and a long time for measurement. Meanwhile, the homogeneous methods does not require washing, reflowing and immobilizing steps. This feature leads to short time measurements. Additionally, homogeneous methods are suitable for small molecular weight targets. Therefore, homogeneous immunoassays such as fluorescence polarization immunoassay (FPIA), particle-enhanced turbidimetric inhibition immunoassay (PETINIA), and cloned enzyme donor immunoassay (CEDIA) are often used for drug detection in samples.9–11 We have reported FPIAs using microdevices.12–15 Although we succeeded in showing the effectiveness of TDM implemented with a microdevice, measurement of drug concentration in whole blood was not accomplished. It remains a major challenge.

Among homogeneous methods, CEDIA has been widely used recently. It was developed by Henderson et al.16Fig. 1 shows a schematic illustration of CEDIA. By recombinant DNA manipulation, the bacterial enzyme β-galactosidase is genetically divided into two inactive fragments: an enzyme acceptor (EA) and an enzyme donor (ED). The complementation of EA and ED forms an active enzyme. The attachment of an analyte to ED is insensitive to the ability of ED to associate with EA. Analyte–ED conjugate binding to an antibody does not associate with EA and does not form an enzyme. An analyte in a sample competes with analyte–ED conjugate for a limited number of antibody binding sites, making an analyte–ED conjugate available for enzyme formation. The amount of active enzyme formed is proportional to the concentration of an analyte.


image file: c8lc01105b-f1.tif
Fig. 1 Schematic illustration of cloned enzyme donor immunoassay (CEDIA).

In this study, we developed a microdevice that was capable of TDM with whole blood in a facile analysis. As the model analyte for TDM, we used theophylline which is a therapeutic agent for bronchial asthma. The detection reagent was stored on the device by lyophilization and theophylline in the blood was detected by CEDIA. Usually, for the analysis of whole blood, pretreatments such as blood cell separation and quantitative sampling of plasma are required. However, these take a long time and their operation is complicated. In this study, we were able to perform sample pretreatment and detection by incorporating a plasma separation membrane into the device. As a result, we succeeded in providing the microdevice for simple and rapid TDM.

Experimental

Chemicals

Acetone, isopropanol, dimethyl sulfoxide (DMSO), casein from milk, phosphate buffered saline (−) (PBS) and fluorescein di-β-D-galactopyranoside (FDG) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). SYLGARD 184 Silicone Elastomer Kit was purchased from Dow Corning Toray (Tokyo, Japan). SU-8 3050 and SU-8 developer were purchased from Microchem Corporation (Newton, MA). The CEDIA Theophylline II Kit was purchased from Microgenics Corporation (Fremont, CA). The CEDIA Theophylline II Kit included reagents 1 and 2. Reagent 1 was a mixture of EA and antibody and reagent 2 was a mixture of theophylline–ED conjugate and 2-(N-morpholino)ethane sulfonic acid (MES) buffer (reagent 2 was a custom-made reagent). Human serum was purchased from Gemini Bio-Products (Woodland, CA). Human whole blood from a 43 year-old male with sodium heparin anticoagulant was purchased from Cosmo Bio Corporation (Tokyo, Japan). The whole blood sample and wastes contaminated by the blood were disposed according the regulations of the Hokkaido University.

Fabrication of devices

A silicon wafer (3 inches × 3 inches, Silicon Sense, Nashua, NH) was washed with acetone and isopropanol. The wafer was dried on a hot plate at 200 °C for 5 min. SU-8 3050 was dropped onto the wafer and coated at 3000 rpm for 45 s using a spin coater (MS-B200, Mikasa Co. Ltd., Tokyo, Japan). Then pre-baking was carried out by heating the coated wafer on a hot plate at 95 °C for 7 min. A high-resolution chromium mask having a pattern of the microchannel was put on the wafer which was irradiated with UV light of 20 mW cm−2 for 15 s with an exposure machine (Karl Suss MJB 3, Class One Equipment, Garching, Germany). Next, the wafer was heated on a hot plate at 95 °C for 9 min before being immersed in SU-8 developer for 6 min to remove uncured photohardenable resin. Finally, the wafer was washed with isopropanol and dried using an air compressor. The mold having a pattern of the microchannel was completed.

A 10[thin space (1/6-em)]:[thin space (1/6-em)]1 mixture of PDMS oligomer and cross-linking agent (SYLGARD 184), which had been degassed under vacuum, was poured onto the mold that had been placed in a plastic Petri dish. The PDMS-containing mixture was cured by heating on a hot plate at 95 °C for 2 h. Then, the PDMS with microchannel shape transferred was peeled off from the mold. This PDMS was cut into a rectangle (18 mm × 28 mm) and inlet (2.5 mm diameter) and outlet (0.35 mm diameter) were made using a punch (Harris Uni-Core, Ted Pella, Inc., Redding, CA).

A 24 mm × 32 mm cover glass (Matsunami Glass, Osaka, Japan) and a prepared PDMS chip were washed with isopropanol and dried with an air compressor. These were bonded together to complete the microdevice (Fig. 2(a)). This microdevice has a microchannel including the straight channel and four reaction chambers for enclosing the detection reagent. The size of the straight channel and a reaction chamber are 200 μm (width) × 12 mm (height) × 50 μm (depth) and 100 μm (width) × 700 μm (height) × 50 μm (depth). This microdevice has a microchannel including four reaction chambers for enclosing the detection reagent. It is possible to perform the same measurement four times by a single injection of a sample. For the microdevice for whole blood analysis, a plasma separation membrane (Nihon Pall, Tokyo, Japan) was shaped into a circle with a diameter of 2.5 mm and attached to the inlet of the device (Fig. 2(b)).


image file: c8lc01105b-f2.tif
Fig. 2 (a) Photograph of the microdevice and schematic illustration of the microchannel. (b) Photograph of the microdevice for whole blood analysis.

Preparation for assay on the microdevice

In the commercially available CEDIA Theophylline II Kit, the concentration of theophylline is measured by the hydrolysis reaction of the chromogenic substrate chlorophenol red-β-galactopyranoside (CPRG). In this study, we measured the concentration of theophylline by customizing reagent 2 (it did not contain CPRG) and detecting the fluorescent substrate FDG by a hydrolysis reaction.17 The following reagents were mixed to prepare the detection reagent. Detection reagent was composed of CEDIA-Theophylline II R1 (50.0 μL), CEDIA-Theophylline II R2 (without CPRG) (48.6 μL) and 0.1 M FDG in DMSO (1.35 μL).

The device was placed in a vacuum desiccator and left for 5 min to remove air contained in the PDMS. PBS solution containing 0.05 wt% casein (3 μL) was dropped into the inlet of the device and introduced into the microchannel with a syringe. After incubation for 30 min, air was introduced using a syringe to remove the casein solution. Thereafter, PBS (3 μL) was introduced and incubation was continued for 2 min. After washing with PBS, this device was placed in the vacuum desiccator and left for 20 min.

After removing air contained the device in the vacuum desiccator, the detection reagent (2 μL) was dropped into the inlet and left for 1 min. The detection reagent was introduced into the device by capillary action. Then air was introduced and the precise volume of the reagent was left in the reaction chambers. Following this, the device was placed in a deep freezer at −80 °C for 1 h. Next, it was lyophilized overnight in a freeze dryer (FDS-1000, EYELA, Tokyo, Japan). The temperature and pressure of the freeze dryer were set to be not higher than −85 °C and 4.0 Pa.

Theophylline assay

For the model serum sample, theophylline, as model analyte, was dissolved in human serum at different concentrations. The final concentration of theophylline was adjusted to 0, 5, 10 or 20 μg mL−1. For the model whole blood sample, 4 mg mL−1 theophylline in PBS solution was dissolved in commercial human whole blood. The final concentration of theophylline was adjusted to 10 μg mL−1 in whole blood sample.

Devices were taken from the freeze dryer at the time of use. Since the device was stored at low pressure in the freeze dryer, a sample could be introduced into the reaction chambers in the device immediately.

The sample (1 μL) was dropped into the inlet. We confirmed that the sample was introduced into the reaction chambers by capillary force. The time at which the sample reached the reaction chamber among the four that was the furthest from the inlet was taken as the reaction start time. When the reaction time reached 10 s, the device was placed on a temperature controller that was heated to 30 °C. When the reaction time reached 1 min, air was injected to take precise volumes of the sample into the device. When the reaction time was 2 min, the device was irradiated with excitation light and a fluorescent image was taken. Fluorescence images were similarly taken at reaction times of 3 min, 4 min and 5 min. Based on the captured fluorescence images, the fluorescence intensity was defined as the average value of the fluorescence signals from all pixels in each reaction chamber.

Results and discussion

Operation test of devices

In order to realize the measurement of the blood sample by only manual operation, we designed a device that has a precise amount of detection reagent enclosed, in other words, it is prefilled. Previous researches has suggested that quantitative dispensing of reagent in the device was performed by combining microchannels of different width that generate a pressure difference.18,19 By adopting this principle, quantitative dispensing could be accomplished by simply introducing air. We designed the microdevice having a wide microchannel (width: 200 μm) and narrow reaction chambers (width: 100 μm) for quantitative dispensing. However, a problem was that bubbles formed in the sample and they affected reproducibility of dispensing of the sample. To solve this problem, air bubbles generated when the sample was introduced into a reaction chamber were removed by utilizing air permeability of PDMS.20,21 A reagent was introduced with a syringe, then air was introduced to take a precise amount of this reagent to its reaction chambers (Fig. 3(a)). After this prefilling, the CEDIA reaction was performed simply by introducing the sample in the same manner (Fig. 3(b)).
image file: c8lc01105b-f3.tif
Fig. 3 Schematic illustration of the microdevice. (a) Prefilling detection reagent procedure: ① reagent injection, ② air injection and ③ lyophilization. (b) Fluorescence assay procedure: ① sample injection, ② air injection and ③ fluorescence assay.

Water was dropped into the inlet of the device for evaluation of quantitativity and reproducibility of dispensing water. The bright field image was taken with a microscope, and the number of pixels was analyzed (Fig. 4). The error (%) was calculated by the ratio of the number of pixels of the field filled with water and the blank field in each reaction chamber. An evaluation was carried out using 13 devices. Each device had four reaction chambers, therefore evaluation was conducted at 52 reaction chambers in total. As a result, the variation coefficient (CV) of the volume of the water between 52 reaction chambers was 1.1% (Fig. 4(a)). CV of the volume of the water between 13 devices was 0.71% (Fig. 4(b)). According to Fig. 4(a), the position of the reaction field did not correlate with quantitativity. From these above results, we concluded that our designed microdevice has good quantitativity and reproducibility regarding dispensing reagents.


image file: c8lc01105b-f4.tif
Fig. 4 Evaluation of quantitativity and reproducibility of dispensing water. (a) Comparison between reaction chambers (13 devices × 4 reaction chambers). (b) Comparison between devices (13 devices).

Next, the detection reagent was enclosed in the device and the device was lyophilized. After lyophilization, we tested that how long it took to introduce the model serum sample (Fig. 5). The time at which the sample was introduced was defined as 0 s. The black areas of the reaction chambers in Fig. 5(a) are the lyophilized detection reagents. The reagent was stored quantitatively in the reaction chambers without bubble contamination due to air permeability of degassed PDMS. Fig. 5(b)–(d) show that the introduced model serum sample gradually invaded the reaction chambers. The sample completely filled the reaction chamber in 44 s. Although the time until the introduction was completed had a slight variation between the devices, the introduction of the sample was completed within 50 s in all devices (ESI Movie S1).


image file: c8lc01105b-f5.tif
Fig. 5 Photographs of the reaction chambers including lyophilized detection reagents obtained at different times after introducing the sample: (a) 0 s (b) 10 s (c) 30 s (d) 44 s.

Analysis of theophylline in serum

The theophylline in serum was introduced into the device that had the lyophilized enclosed detection reagent and the fluorescence intensity was measured (Fig. 6). Incubation was carried out for a certain period of time after introduction of the sample, and then the fluorescence intensity was measured. When FDG, a non-fluorescent molecule, is hydrolyzed by β-galactosidase, one galactose molecule is desorbed and becomes fluorescein monogalactoside (FMG). When further hydrolyzed, another galactose molecule is desorbed, and it becomes fluorescent fluorescein. Since the amount of fluorescein is proportional to the amount of β-galactosidase, it can be used for the detection of theophylline. As shown in Fig. 6(a), an increase in fluorescence intensities with each concentration increased over time. The calibration curve of theophylline was prepared at incubation times of 4 min and 5 min (Fig. 6(b) and (c)). The fluorescence intensity sharply increased in the low concentration region and converged to a constant value in the high concentration region. We previously reported that the calibration curve of theophylline with CEDIA Theophylline II Kit was linear from 0 μg mL−1 to 40 μg mL−1 of theophylline.17 There is a possibility that the influence of reagent concentration, antibody or enzyme activity may be affected by lyophilization. Therefore, by examining the reagent concentration in detail, we considered that the linear region could be expanded in the calibration curve. The limit of detection (LOD) was calculated as 0.64 μg mL−1 when the incubation time was 4 min and LOD was 0.38 μg mL−1 at 5 min. In general, the therapeutically effective concentration range of theophylline was 5–15 μg mL−1, and the obtained detection limit was considered to be sufficient for targeting theophylline by TDM.
image file: c8lc01105b-f6.tif
Fig. 6 (a) Plots of fluorescence intensity vs. reaction time at various concentrations of theophylline in serum. (b) and (c) Calibration curves for theophylline in serum. Reaction time: (b) 4 min and (c) 5 min. Error bars are standard deviations in the four reaction chambers of the same device (N = 3).

Analysis of theophylline in whole blood

A plasma separation membrane was put in the inlet of the device, and theophylline in the whole blood sample was measured. This sample was not subjected to pretreatment such as anticoagulant use. We confirmed that blood cell components could be separated and only plasma would be introduced into the device. We measured the fluorescence intensity of the introduced sample in the same manner as in the serum model sample (Fig. 7). Increasing fluorescence intensity was confirmed just as for the serum model sample and as incubation time increased, LODs were 1.2 μg mL−1 at 4 min and 0.73 μg mL−1 at 5 min. The theophylline concentration in whole blood could be satisfactorily analyzed with an incubation time of 4 min.
image file: c8lc01105b-f7.tif
Fig. 7 (a) Plots of fluorescence intensity vs. reaction time at various concentrations of theophylline in whole blood. (b) and (c) Calibration curves for theophylline in whole blood. Reaction time: (b) 4 min and (c) 5 min.

Fig. 8 shows the results of evaluation of variation between reaction chambers and devices. Fluorescence intensities of 10 μg mL−1 theophylline in whole blood at a reaction time of 4 min were compared. CV between different reaction chambers in the same device was 4.9% (Fig. 8(a)) and in different devices CV was 1.7% (Fig. 8(b)). Our device has sufficient accuracy for use in TDM.


image file: c8lc01105b-f8.tif
Fig. 8 Evaluation of variation between (a) reaction chambers and (b) devices. Concentration of theophylline in whole blood was 10 μg mL−1 and reaction time was 4 min.

Finally, the material cost per one assay was calculated. The total material cost of the devices and reagents used in this study was ∼$6.70. The cost of FDG accounts for most of this price. We used the FDG for convenience with the equipment. However, the material cost can be reduced to ∼$0.30 if detection can be done by absorption of CPRG contained in the commercially available CEDIA-Theophylline II Kit. This cost is low enough for POCT use. From these results, we confirmed the successful development of a microdevice that can measure theophylline in whole blood samples immediately after blood collection quickly, inexpensively and facilely.

Conclusions

In this study, we developed a microdevice that can measure blood drug concentration conveniently and quickly for POCT by TDM. We showed that our device can easily provide quantitative dispensing of liquid by just manual operation of a syringe. In addition, we confirmed the quantitativity and reproducibility of dispensing liquid were good. For the theophylline detection, CEDIA was used. LODs of theophylline were 0.38 μg mL−1 in the serum model and 0.73 μg mL−1 in the whole blood model samples. These LODs are adequate for theophylline assay in TDM. The required volume of the whole blood sample was 2 μL and the measurement time was 4 min. Therefore, we succeeded in developing a microdevice that can measure theophylline in whole blood samples immediately, inexpensively and facilely after blood collection. By optimizing the concentration of the detection reagent, we expect to improve the device sensitivity.

Finally, to put our device into practical use, it is necessary to consider preservability, that is shelf-life of the device before use. In order to improve shelf-live, we think that using a well-known method such as adding a sugar like trehalose or sucrose is needed.22 In addition, the device must be packed in a vacuum to keep its permeability.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We thank Dr. Rueyming Loor of Microgenics Corporation for providing CEDIA kits. We also acknowledge Prof. Tomoya Tachi of Gifu Pharmaceutical University for valuable discussion.

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Footnotes

Electronic supplementary information (ESI) available. See DOI: 10.1039/c8lc01105b
Equally contributing author.

This journal is © The Royal Society of Chemistry 2019