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Sensors and Materials, Vol. 34, No. 3 (2022) 971–985
MYU Tokyo
S & M 2867

Development of Portable Fluorescence Microplate Reader
Equipped with Indium Tin Oxide Glass Heaterfor Loop-mediated Isothermal Amplification
Ryo Ishii,1 Kazuhiro Morioka,2* Takuya Mizumoto,1 Natsumi Yamasaki,1
Akihide Hemmi,3 Atsushi Shoji,2 Hiroya Murakami,4 Norio Teshima,4Tomonari Umemura,5 Katsumi Uchiyama,1 and Hizuru Nakajima1**
1Department of Applied Chemistry, Graduate School of Urban Environmental Sciences,

Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, Tokyo 192-0397, Japan
2Department of Biomedical Analysis, School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
3Mebius Advanced Technology Ltd., 3-31-6 Nishiogi-kita, Suginami-ku, Tokyo 167-0042, Japan
4Department of Applied Chemistry, Aichi Institute of Technology,
1247 Yachigusa, Yakusa-cho, Toyota, Aichi 470-0392, Japan
5School of Life Sciences, Tokyo University of Pharmacy and Life Sciences,1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan(Received September 3, 2021; accepted November 18, 2021; online published January 5, 2022)

Keywords: fluorescence microplate reader, LAMP, ITO glass heater, on-site nucleic acid test, variety
discrimination

We developed a portable fluorescence microplate reader equipped with an indium tin oxide(ITO) glass heater for nucleic acid testing based on loop-mediated isothermal amplification(LAMP). The components used for detection (e.g., LEDs, optical filters, the microwell plate, theITO glass heater, and photodiodes) are arranged in parallel on a straight line. This opticalplacement allows us to simultaneously heat nine sample solutions and monitor the fluorescenceintensity amplified by the LAMP reaction despite the analysis instrument being handheld. This
simultaneous monitoring was demonstrated by the LAMP reaction using positive and negative
controls. In addition, the microplate reader was applied to the variety discrimination of rice, andKoshihikari rice was successfully discriminated from four types of milled rice using the
microplate reader. The developed microplate reader will be useful for on-site nucleic acid testing
because it can be operated using batteries and is portable.

1.

Introduction

Recently, portable nucleic acid test systems for on-site diagnosis have attracted a lot of
attention.(1–3) Carrying out nucleic acid testing in the field is useful for point-of-care testing,(4)environmental analysis,(5) and food safety testing(6) because the result can be obtained rapidly.
To miniaturize and reduce the cost of nucleic acid test systems, the design of the detection andheating parts is very important. Although the real-time polymerase chain reaction (PCR)(7) is the*Corresponding author: e-mail: kmorioka@toyaku.ac.jp**Corresponding author: e-mail: nakajima-hizuru@tmu.ac.jp

https://doi.org/10.18494/SAM.2022.3618

ISSN 0914-4935 © MYU K.K.
https://myukk.org/

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gold standard method among nucleic acid amplification techniques, the analysis requires adevice for multistep heating and a highly sensitive detection system for measuring thefluorescence signal of dye molecules existing at low concentrations. Various types of miniaturereal-time PCR systems have been developed,(8–10) but the systems are large and complicated inmany cases. In addition, the real-time PCR requires a long analysis time for the heater control
cycle as well as for PCR amplification.
Loop-mediated isothermal amplification (LAMP),(11) which is one of the nucleic acidamplification methods, is very useful for on-site nucleic acid testing since it is capable of rapid
nucleic acid amplification by isothermal heating. LAMP can be adapted to various detection
methods [e.g., turbidimetry(12) and colorimetry(13)] as well as to the fluorescence detection
method,(14,15) and LAMP enables us to discriminate the presence or absence of gene
amplification visually.(16–18) Moreover, isothermal heating enables us to simplify the system
configuration. Owing to these advantages, many research groups have studied LAMP systems
based on visual detection for on-site nucleic acid testing.(19–21) Although visual discrimination is
simple and does not require a detection device, the obtained result is semi-quantitative. On theother hand, real-time signal monitoring during the LAMP reaction(22) is useful for rapidly
finding abnormalities caused by contamination as well as for the quantitation of nucleic acid.
More recently, some detection devices have been developed for real-time LAMP reaction
monitoring, such as a small detection system using an LED and a photodiode (PD),(22,23) a tube
scanner,(24) and a smartphone-based detection system.(25) These detection devices were not only
small but also easy to use and had a high detection sensitivity and a low fabrication cost. On theother hand, for the heating part of the LAMP system, downsizing, weight saving, low electric
power consumption, and temperature stability are required. Various types of heaters [e.g., film
heaters(26,27) and heating coils(28)] have been used for portable LAMP systems. The performanceof these heaters is sufficient to perform the LAMP reaction, but the optical arrangement of thedetection system is limited because of the opaque heater material.
An indium tin oxide (ITO) glass heater(29) has advantages such as easy fabrication at a low
cost and heating with low electrical power, similarly to other heaters used for portable analysis
systems. Moreover, several ITO heaters can be fabricated on a single glass plate by chemical
etching.(30–32) However, the most noteworthy feature of ITO heaters is the high opticaltransmittance. Since an ITO heater can transmit light in the visible and near-IR regions, it isuseful for measuring a heated substance using optical detection methods. Because of thesecharacteristics, ITO heaters have been widely used in biological analyses such as cell analysis(33)and hybridization.(34) Several research groups have applied an ITO heater to gene testing basedon PCR.(30–32) Wu et al. developed a thermostat chip with an ITO glass substrate for static
PCR.(35) The fabricated system including the thermostat chip was successfully applied to the insitu real-time monitoring of the fluorescence of SYBR Green Ⅰ during PCR. However, it isdifficult to apply the system to on-site gene testing because it requires large and expensive
peripheral instruments, i.e., a fluorescence microscope and a photomultiplier for fluorescence
detection. Until now, an ITO glass heater has not been applied to a portable simultaneous genetesting device for the real-time fluorescence monitoring of the LAMP reaction. The main reason
for this is that a fluorescence detector commonly requires an optical system, in which a lightSensors and Materials, Vol. 34, No. 3 (2022)

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source and a photodetector are vertically arranged, increasing the size and complexity of thedetection system.
Previously, we developed a portable fluorescence microplate reader for enzyme-linked
immunosorbent assay (ELISA).(36) The optical components of the microplate reader, i.e., lightsources, two optical filters, and photodetectors, were arranged in parallel on a straight line. This
optical system enabled us to detect the fluorescence signal in nine microwells simultaneously.
The microplate reader was successfully applied to detect immunoglobulin A (IgA) in humansaliva.
Herein, we developed a portable fluorescence microplate reader equipped with an ITO glass
heater for LAMP. The flat heater with high transparency and the detection components werearranged in parallel to monitor fluorescence intensity changes in nine wells during the LAMP
reaction simultaneously. The heating and detection performance characteristics of the microplate
reader were precisely evaluated and then the microplate reader was applied to the variety
discrimination of rice based on LAMP.

2.

Materials and Methods

2.1

Reagents and materials

Water purified by a Direct-Q system (Nihon Millipore, Tokyo, Japan) was used for sample
preparation. All chemical reagents used in this work were of analytical grade unless otherwise
stated. The polydimethylsiloxane (PDMS) base compound and curing reagent (DOWSIL™
SILPOT 184 W/C) were purchased from Dow Corning Toray Co., Ltd. (Tokyo, Japan). Carbon
black (K-COLOR-BK-02) was purchased from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Ag/
AgCl paste was purchased from Gwent Group Co., Ltd. (Gwent, UK). Calcein and hydrochloric
acid were purchased from Kanto Chemical Co., Ltd. (Tokyo, Japan). 2-Amino-2-hydroxymethyl1,3-propanediol (Tris) was purchased from Fujifilm Wako Pure Chemical Co., Ltd. (Osaka,
Japan). Chlorine bleach was purchased from Kao Co., Ltd. (Tokyo, Japan). The Koshihikari
LAMP discrimination kit containing a LAMP primer, an enzyme reagent,a fluorescence color
reagent, positive and negative controls, ​and sterilized water was purchased from Nippon Gene
Co., Ltd. (Tokyo, Japan).
2.2

Fabrication of nine-well microtiter plate

The mold of the nine-well microtiter plate was fabricated using polyoxymethylene (POM)
plates and cylindrical stainless pins (φ 4.0 mm × H 10 mm). A baseplate (L 50 mm × W 50 mm ×
H 10 mm) having nine holes (φ 4.0 mm × D 5 mm) and a square convex portion (L 30 mm × W
30 mm × H 3 mm), and a mold form (L 50 mm × W 50 mm × H 8 mm) having a square throughhole (L 30 mm × W 30 mm) were formed using a milling machine (KitMill RD420,
ORIGINALMIND Co., Ltd., Nagano, Japan). The cylindrical stainless pins were inserted into
each hole of the baseplate after the bottom surface of the pins was polished with a liquid abrasive(Pikal, Nihon Maryo Kogyo Co., Ltd., Tokyo, Japan). The mold of the nine-well microtiter plate974

Sensors and Materials, Vol. 34, No. 3 (2022)

was fabricated by fitting the mold form into the baseplate and then fixed by taping the outside of
the plates with an adhesive tape.
The nine-well microtiter plate was made from PDMS and carbon black paste. The fabrication
procedure for the nine-well microtiter plate is shown in Fig. 1(a). The PDMS base compound andcuring agent were mixed at a weight ratio of 10 to 1. The PDMS mixture and carbon black paste
were mixed at a weight ratio of 19 to 1, and this black mixture was degassed using a vacuum
pump (G-25SA, ULVAC, Inc., Kanagawa, Japan). The degassed black mixture was poured into
the mold and the mold was covered with an acrylic block (50 × 50 × 50 mm3, Mebius Advanced
Technology Ltd., Tokyo, Japan). The mold containing the black mixture was heated at 70 ℃ for
1.5 h in an oven (ST100, ESPEC Co., Ltd., Osaka, Japan) to polymerize the PDMS. After that,the cured black PDMS plate was peeled off from the mold, and nine through holes (φ 3.5 mm)
were formed on the black PDMS plate using a biopsy punch (BP-35F, Kai Industries Co., Ltd.,Tokyo, Japan). This plate was returned to the mold and then the PDMS mixture without carbon
black was poured on the mold. The mold was covered with the acrylic block and then cured at 70℃ for 1.5 h. After the PDMS was cured, the finished nine-well microtiter plate was peeled offfrom the mold. The design and photograph of the nine-well microtiter plate are shown in Figs.
1(b) and 2(b), respectively. Since it was difficult to completely remove amplicon by washing thewells, the nine-well microtiter plate was not reused. Before use, the microtiter plate was washedwith 1.0% (v/v) chlorine bleach aqueous solution to prevent contamination.
2.3

Fabrication of ITO transparent heater

A piece of ITO transparent glass (100 × 100 × 1.1 mm3, No. 1001, Geomatec Co., Ltd., Tokyo,
Japan) was cut to 20 × 40 mm2 using a glass cutter, then cleaned with ultrapure water. Afterdrying, two wires were fixed on both ends of the ITO glass with Ag/AgCl paste, and the paste
was cured at 150 ℃ for 30 min. The performance of the ITO glass heater was evaluated using athermal camera (PI400i, Argo Co., Ltd., Berlin, Germany).

Fig. 1. (Color online) Nine-well microtiter plate made from PDMS and carbon black. (a) Fabrication procedure forXthe plate. (b) Design of the plate.

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2.4

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Development of portable fluorescence microplate reader

The optical design and photograph of the portable fluorescence microplate reader developedin this work are shown in Fig. 2. The optical system of the plate reader consisted of nine highbrightness blue LEDs (OSUB5161P, OptoSupply Ltd., N.T., Hong Kong), a short-pass filter (SPF)(SVX490, Asahi Spectra Co., Ltd., Tokyo, Japan), a long-pass filter (LPF) (LVX530, Asahi
Spectra), the ITO glass heater, two perforated light-shielding plates, and nine PDs(TEMD7000X01, Vishay Intertechnology, Inc., PA, USA). The detection of isothermal gene
amplification using the microplate reader was performed as follows. The nine-well microtiterplate was set in the plate reader and the sample solutions in the nine microwells on the microtiterplate were heated using the ITO glass heater until the measurement was finished. The excitation
light beams from the LEDs (peak emission wavelength: 467 nm) were passed through the SPF(cut-off wavelength: 490 nm) and ITO glass heater, and then irradiated the solutions in the
microwells. The fluorescence of calcein generated in the microwells was passed through the LPF(cut-on wavelength: 530 nm) and reached the PDs. The fluorescence was converted tophotocurrent by the PDs. The photocurrent was converted to an analog voltage and amplified bya current/voltage (I/V) conversion amplifier (Mebius Advanced Technology Ltd., Tokyo, Japan).
The voltage was converted to a digital value by an analog/digital (A/D) converter (ADS1115,
Adafruit Industries, NY, USA). The digital values were collected by a central processing unit
(CPU) (Arduino Nano 3.0, Gravitech LLC, NV, USA) as measurement data once per second, andthe data were transmitted to a laptop PC by a Bluetooth communication module [Bluefruit EZLink - Bluetooth Serial Link & Arduino Programmer (v 1.3), Adafruit Industries]. The
temperature of the ITO glass surface was monitored with a thermistor (103 JT-025, SEMITEC

Fig. 2. (Color online) Portable microplate reader developed in this work. (a) Optical design of the microplate
reader. (b) Photographs of the microplate reader, nine-well microtiter plate, and photodiodes.

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Co., Ltd., Tokyo, Japan) and heating was controlled using the CPU to a constant temperature bysetting the heater to turn on when the temperature was below 63 ℃. In a result obtained bymeasuring a positive control, the fluorescence increases with time because the amount of thetarget gene is increased by LAMP. The current applied to the LEDs was set to ca. 20 mA. The
plate reader was driven using four 1.2 V batteries (Toshiba Co., Ltd., Tokyo, Japan) and a DC
power supply (Sunhayato Co., Ltd., Tokyo, Japan). The total size and weight of the plate reader
were 130 mm (L) × 80 mm (W) × 80 mm (H) and approximately 400 g, respectively.
2.5

LAMP reaction

The variety discrimination of rice was carried out using the LAMP kit. Using this kit,
Koshihikari rice is identified by discriminating the presence or absence of the amplification of
the region that corresponds to the first intron of resistance gene Pi5-1. Gene testing using the
LAMP kit enables Koshihikari to be distinguished from other breeds. In this experiment, fourtypes of milled rice (Koshihikari, Akitakomachi, Hitomebore, and Yumepirika) were used as
samples. DNA extraction was performed according to the attached manual of the LAMP kit.(37)
A 10 g sample of milled rice was placed in a centrifuge tube and then 19 mL of sterilized waterand 1 mL of 20 × DNA extraction solution No. 1 were added to the tube. After mixing, 500 µLof the supernatant in the tube was transferred to a microtube and then an equal volume of DNA
extraction solution No. 2 was added to the microtube. The solution was mixed and thencentrifuged at 12000 g for 1 min using a centrifuge (HSC-12000, AS ONE Co., Ltd., Osaka,
Japan). The supernatant in the tube was collected in another tube and the solution was diluted
tenfold with sterilized water. The prepared solution was used as a sample solution in thefollowing experiment.
The LAMP reaction in this work was performed using the Koshihikari LAMP discrimination
kit and a fluorescent detection reagent containing calcein and manganese. Twenty-five
microliters of a solution comprising 20.5 µL of the test reagent (No. 1 or No. 2), 1 µL of thefluorescence detection reagent, 1 µL of an enzyme solution, and 2.5 µL of the sample solution
was added to a microwell on the microtiter plate. The microtiter plate was set in the developed
microplate reader and then the amplification was carried out at 63 ℃ for 40 min. During the
LAMP reaction, the fluorescence intensity of the solution in the microwell was monitored in realtime. The breeds of rice were discriminated by comparing the fluorescence intensity of thesample solution with those of the positive and negative controls.

3.

Results and Discussion

3.1

Optical design of fluorescence microplate reader

The fluorescence reagent for LAMP contains calcein. The fluorescence of calcein in thereagent is initially quenched by binding with manganese ions. As the isothermal amplification
reaction proceeds, the generated pyrophosphate removes the manganese ions from calcein, andaccordingly the calcein fluoresces. The fluorescence of calcein increases in the presence ofSensors and Materials, Vol. 34, No. 3 (2022)

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Fig. 3. (Color online) Optical spectra of the reagent and parts of the microplate reader. (a) Excitation spectrum ofcalcein. (b) Emission spectrum of calcein. (c) Excitation spectrum of the blue LED. (d) Transmission spectrum of the
SPF. (e) Transmission spectrum of the LPF. (f) Transmission spectrum of the ITO glass heater. Arrows next to theletters show the corresponding axis.

manganese ions in the reaction mixture. On the basis of this principle, we designed the opticalsystem of the fluorescence microplate reader for detecting the fluorescence of calcein. The
optical spectra of calcein were investigated using a spectrofluorometer (RF-5300PC, Shimadzu
Co., Ltd., Kyoto, Japan) [Figs. 3(a) and 3(b)]. The wavelengths of maximum excitation andemission were 493 and 512 nm, respectively. In the developed microplate reader, the increase inbackground signal intensity due to excitation light was a concern because the LEDs and PDs
were arranged in parallel on a straight line to detect fluorescence in nine microwells
simultaneously.
To overcome this problem, high-brightness blue LEDs having a peak emission wavelength ofless than 512 nm were selected as an excitation light source, and the SPF and LPF were mounted
between the LEDs and the PDs. The emission spectrum of the LED was measured using aspectrometer (Colorcompass MF, AT System, Shizuoka, Japan). The peak emission wavelengthand full width at half maximum of the LED were 467 and 22 nm, respectively [Fig. 3(c)]. Figures
3(d) and 3(e) show the transmission spectra of the filters. The cut-off wavelength of the SPF andthe cut-on wavelength of the LPF were 490 and 530 nm, respectively. The microplate reader withthe filters successfully detected the fluorescence of calcein without a background signal. The
transmittance property of the ITO glass heater was investigated, and the transmittance at 467
nm (i.e., the peak emission wavelength of the LED) was approximately 75% [Fig. 3(f)]. The
result revealed that the fluorescence of calcein can be detected using the plate reader equippedwith the ITO heater without any problems.
3.2

Evaluation of optical crosstalk

To prevent the generation of background noise caused by stray light, the nine-well microtiterplate was fabricated from the PDMS mixture containing carbon black, except for the bottom of
the microwells. Although the opacity of the black PDMS plate containing carbon black was978Sensors and Materials, Vol. 34, No. 3 (2022)

sufficient, optical crosstalk might have occurred because of the short distance between the
microwells (6 mm) and because the fluorescence in the nine microwells was detected using nine
LEDs and nine PDs arranged in close proximity. Thus, the effect of the crosstalk was evaluatedusing calcein solution. A 25 µL portion of 50 µM calcein solution prepared with 50 mM TrisHCl buffer (pH 8.0) (Tris buffer) was added to one of the microwells, whereas a 25 µL portion of
the Tris buffer was added to the other wells, and then the fluorescence intensities in all the
microwells were measured using the developed microplate reader. Figure 4 shows the results ofthe crosstalk evaluation performed for all microwells in turn. Compared with the fluorescenceintensity in the microwell in which calcein solution was added, the fluorescence intensities in theother microwells were very low. This result indicates that the two light-shielding plates with ninethrough holes arranged above and below the LPF prevented the incidence of fluorescenceemitted from the microwell into the PDs corresponding to the other microwells, and thefluorescence in the nine microwells can be detected normally without crosstalk.
3.3

Evaluation of quantitative performance

To evaluate the quantitative performance of the developed microplate reader, calcein solutionsat several concentrations were measured. A 25 μL portion of a calcein solution diluted with theTris buffer was added to the microwells, and the fluorescence intensities were measured using

Fig. 4. (Color online) Fluorescence intensity in nine microwells when the solution containing calcein was added toeach microwell and water was added to the other microwells.

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Fig. 5.

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Calibration curves for calcein.

the microplate reader. The obtained calibration curves for calcein are shown in Fig. 5. The
fluorescence intensity increased with the concentration of calcein and became almost constantfor concentrations above 250 μM. The calibration curves of each well for calcein were linear forconcentrations below 63 μM with correlation coefficients of 0.991–0.999. The limits of detection(LODs), defined as three times the standard deviation of the blank signal, were estimated to be
0.27–1.60 μM. The relative standard deviations (RSDs) (n = 4) in the detectable range (i.e., theconcentration range from the LOD to 63 μM) for each well were below 14.1%. These results
indicate that the fluorescence intensity of calcein solution can be measured quantitatively using
the developed plate reader.
3.4

Heating performance of ITO glass heater

To simultaneously perform the LAMP reaction in all the microwells using the microplate
reader, all microwells must be heated uniformly and stably to the temperature necessary for theisothermal amplification reaction using the ITO glass heater. Thus, the temperature uniformity

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Fig. 6. (Color online) Heating performance of the ITO glass heater. (a) Thermal image of the microtiter plate
heated by the ITO glass heater. (b) Time course of the temperature in the microwells. The line shows the averagetemperature measured in the nine microwells. Heating was carried out for 40 min.

between the microwells was investigated by monitoring the temperatures of water in the nine
microwells heated to 63 ℃ by the ITO glass heater using a thermal camera. The thermal camera
was arranged on the opposite side from the ITO glass heater. The obtained thermogram [Fig.
6(a)] indicates that the ITO glass heater has sufficient performance to heat the microwells
uniformly. From this data, the average temperature of water in the nine microwells wasestimated to be 62.5 ± 2.0 ℃. This result suggests that solutions in all microwells were heated toalmost the set temperature (63 ℃) and that the heating ability is sufficient for the LAMP
reaction.
To carry out the LAMP reaction for the variety discrimination of rice using the plate reader,the sample solution containing the reagents required heating to 63 ℃ for 40 min. Thus, thetemporal temperature stability in the nine microwells was evaluated by measuring thetemperatures of water in the microwells heated to 63 ℃. As shown in Fig. 6(b), the averagetemperature of water in the nine microwells reached 63 ℃ within 95 s from the start of heating,and the temperature remained almost constant for 40 min. After reaching 63 ℃, the averagetemperature of water in the nine microwells for the next 40 min was calculated to be 60.4 ± 0.4℃–65.4 ± 0.2 ℃. In addition, the battery power provided to the heater and peripheral devices
was sufficient for the continuous heating under this condition. These results revealed that theperformance of the ITO glass heater was sufficient to perform the LAMP reaction.
3.5

Variety discrimination of rice

To demonstrate the variety discrimination of real samples extracted from milled rice, a
LAMP test was performed using a thermal cycler and a UV illuminator. A total of 12 PCR tubes
containing different breeds of rice, the positive control, or the negative control, as shown in
Table 1, were heated to 63 ℃ for 40 min using a commercially available thermal cycler (TCLT9610, Blue-Ray Biotech Co., Ltd., Zhongshan Dist., Taipei), and then the fluorescence of theSensors and Materials, Vol. 34, No. 3 (2022)

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Table 1Analytes and reagents used for the variety discrimination of rice.
Sample No.
AnalyteReagent
Reaction
1
No. 1
(+)
Koshihikari
2
No. 2
(−)
3
No. 1
(−)
Akitakomachi
4
No. 2
(+)
5
No. 1
(−)
Hirombore
6
No. 2
(+)
7
No. 1
(−)
Yumepirika
8
No. 2
(+)
9
No. 1
(+)
Positive control
10
No. 2
(+)
11
No. 1
(−)
Negative control
12
No. 2
(−)

solutions was visually observed by irradiating UV light to the solutions using a UV illuminator(SLUV- 4, AS ONE Co., Ltd., Osaka, Japan).
When test reagent No. 1 was used, the solutions containing the positive control or Koshihikari
emitted fluorescence, whereas no fluorescence was observed in the solutions containing thenegative control or the other types of rice. On the other hand, when test reagent No. 2 was used,
the solutions containing the positive control or the rice except for Koshihikari emitted
fluorescence, whereas no fluorescence was observed in the solutions containing the negative
control or Koshihikari. These results were identical with those in the manual.(37)
To demonstrate that the developed microplate reader can be used for the LAMP reaction, the
LAMP amplification of the positive and negative controls was carried out in the nine microwells
using the developed microplate reader. The time course of the fluorescence intensities during the
LAMP amplification is shown in Fig. 7. Specific differences in fluorescence intensity between
the positive and negative controls were observed in all microwells. This result revealed that thedeveloped microplate reader can be used to heat the solution in the microwells and that thefluorescence intensity in all microwells can be monitored in real time during the LAMP reaction
using the developed microplate reader. The time until the fluorescence intensity started toincrease rapidly was different in each microwell. A possible reason for this is that the temperaturein each microwell and the sensitivity of each PD were slightly different. The performance of thedeveloped microplate reader was sufficient to discriminate the positive and negative controls.
However, for the simultaneous determination of DNA, the preparation of calibration curves foreach microwell was required since the heating and detection performance characteristics weredifferent in each microwell.
In the variety discrimination of rice, a decision of the presence or absence of a specific genesequence is only required. Therefore, the performance of the developed microplate reader issufficient for the variety discrimination of rice. The variety discrimination of rice was carriedout using the developed microplate reader. The time courses of the fluorescence intensity in the
microwells for four rice samples, the positive control, and the negative control are shown in Fig.
8(a). When test reagent No. 1 was used, the fluorescence intensity of the solution containing

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Fig. 7. (Color online) Time courses of the fluorescence intensity of the solution containing the positive or negative
control in the nine microwells during the LAMP reaction.

Koshihikari rapidly increased from 17 to 30 min after the start of the LAMP reaction, whereas
the fluorescence intensity of the solutions containing the other types of rice did not change
markedly. On the other hand, when test reagent No. 2 was used, the fluorescence intensity of thethree solutions containing Akitakomachi, Hitomebore, and Yumepirika markedly increased,
whereas that of the solution containing Koshihikari hardly changed. A comparison of thefluorescence intensities obtained 40 min after starting the amplification is shown in Fig. 8(b).
From the comparison of the fluorescence intensities of the sample solutions, the positive control,
and the negative control, it was concluded that the LAMP reaction using Koshihikari waspositive in test reagent No. 1 and negative in test reagent No.2. For the other samples, opposite
results were obtained. These results agree with those in the manual(37) and previous visual
examination.
As described above, the microplate reader developed in this work was successfully used in
the LAMP reaction using DNA extracted from real samples. The microplate reader will be
useful for on-site gene testing in various fields since it is handheld and portable.

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Fig. 8. (Color online) Results of the variety discrimination of rice. (a) Time courses of the fluorescence intensity ofthe sample solutions in the microwells during the LAMP reaction. (b) Comparison of the fluorescence intensities at
40 min.

4.

Conclusions

In this study, a microplate reader equipped with a flat ITO glass heater with high opticaltransparency was developed and the simultaneous monitoring of the LAMP reaction based on
fluorescence detection was demonstrated. The combination of the transparent ITO glass heaterand the detection system using LEDs and PDs enabled the device configuration to be simplified
and the analysis system to be miniaturized. This system is also suitable for other analysis
methods (for example, other isothermal gene amplifications, hybridization, cell analysis, andmeasurements utilizing enzyme reactions).
In addition, the developed system is expected to be applied to the quantification of DNA.
Using the present microplate reader, it is difficult to directly compare the fluorescence intensities
in the different microwells because of small differences in the sensitivity of the PDs, thewavelengths and intensities of the LEDs, and the temperature in each microwell. However, thequantitation of DNA with the developed plate reader might be achieved in the future by adjusting
the detection sensitivity and heating temperature at each well. Although the developed

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microplate reader was only used for the variety discrimination of rice in this study, it has thepotential to be applied to various gene tests, such as infectious disease diagnosis, microorganism
tests, and the detection of allergen substances. Since the microplate reader is handheld and canbe operated using batteries, it is expected to be used in field tests when a disaster occurs.

Acknowledgments
This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI(17K14504, 18H02009, 19H02751, 21H01968, 21H03578, 21K18985) and Tokyo Metropolitan
Government.

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