AMPLIFIED QCM-CHIP FOR DETECTION OF VIRAL RNA

BY NANOPARTICLES Shuichiro Kagiyama1, Taisuke Masuda1*, Kunihiro Kaihatsu2,

Nobuo Kato2 and Fumihito Arai1 1Nagoya University, Japan 2Osaka University, Japan

ABSTRACT

This paper presents a method of detecting viral RNA using Quartz Crystal Microbalance (QCM). We

fabricated microfluidic chip with QCM and immobilized Peptide Nucleic Acid (PNA) on the surface of the

electrode of the QCM to capture viral RNA specifically. Therefore, we used the gold nanoparticles

conjugated anti-nucleoprotein antibody to increase sensing mass and improve sensing limit of QCM. As a

result, we succeeded in detecting viral RNA (5 x 103 [pfu]). And we also confirmed expectation of more

amplitude and reduction in loss by magnetic nanoparticles.

KEYWORDS: QCM, Microfluidic chip, Virus detection, Nanoparticle amplification

INTRODUCTION

For diagnosing the viral infectious diseases quickly and accurately, detection method of infectious viral

RNA has been developed. However, it is necessary to purify the virus and extract the viral RNA in the

sample as the pretreatments before detection of infectious viral RNA and it takes a long time to complete

such pretreatments. Previously, we proposed the microfluidic chip for virus purification and enrichment

and extraction the viral RNA [1]. Then, the purpose of this study is the establishment of detection method

which can be implemented in the previous microfluidic chip.

Then QCM is used for detection. Quartz crystal has piezoelectric characteristics and generates periodic

signal with high stability based on resonance. Binding of the soluble molecule causes a frequency shift in

the resonance frequency of the quartz crystal. By using QCM, it is possible to detect viral RNA rapidly.

THEORY

Signal amplification would be one of the major approaches for designing and developing highly

sensitive QCM [2]. Figure 1 shows the concept of experiment with nanoparticles. We made microfluidic

channel on QCM and immobilized PNA on the specificity. PNA is an artificially synthesized nucleic acid

of which sequence is designable so that the PNA can recognize specific sequence of RNA. The purified

influenza viral RNA are introduced into the channel and are captured by PNA. Here, the nanoparticles

conjugated anti-nucleoprotein antibody are introduced into the channel so as to increase sensing mass. The

target virus genome is captured by PNA and the accompanying viral nucleoprotein (NP) is labeled with

anti-NP antibodies in order to obtain the sensitization action of QCM.

Fig 1: Schematic illustration of the our microfluidic chip concept of to detect viral RNA amplified with nanoparticle.

1

RNA

Nucleoprotein (NP)

Quartz Crystal Microbalance (QCM)

PNA

PDMS

Microfluidic

channel

2 PDMS

Nanoparticle

NP antibody

Quartz Crystal Microbalance (QCM)

Microfluidic

channel

879978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA

Then, we proposed to use two kinds of nanoparticles; gold nanoparticles and magnetic nanoparticles.

The diameter of the gold nanoparticle is about 40 nm. The weight is around 6.5 x 10-16 g, that is about 540

times as heavy as viral RNA. The diameter of the magnetic nanoparticle is about 100 nm or less. The density

is a quarter of the gold nanoparticle, but the collection rate will be improved.

Figure 2 (a) shows the photograph of QCM-chip. The microfluidic channel made of PDMS is combined

with QCM directly. QCM-chip is fixed directly on the oscillation circuit substrate. Figure 2 (b) shows the

design of our microfluidic chip. The fabricated bifacial electrodes were 3 mm in diameter. The sensitivity

is about -125 pg/Hz calculated by Sauerbrey equation [3].

EXPERIMENTAL

Figure 3(a) shows the schematic diagram of experimental setup. The system was composed of a power

supply (IPS4303, ISO THECH corporation), frequency counter (53230A, Aglient Technologies

corporation) and oscillation circuit. Figure 3(b) shows the oscillation circuit.

The experimental procedure was as follows. We set five conditions; (1) introducing viral RNA only

(104 pfu), (2) introducing the gold nanoparticles only, (3) introducing the gold nanoparticles after

introducing viral RNA (5 x 103 pfu), (4) introducing the gold nanoparticles after introducing viral RNA

(104 pfu), lastly (5) introducing magnetic nanoparticles only. Condition 5 was as the basic experiment of

magnetic nanoparticles.

RESULTS AND DISCUSSION

Figure 4 and 5 shows the results of mass measurement experiment. From the results of figure 4, the graphs

of condition 1 and condition 2 show little frequency drop. On the other hand, the graphs of condition 3 and

condition 4 show significant frequency drop, since gold nanoparticles are introduced after introduction of viral

RNA. In comparison with viral RNA, the frequency change of 104 pfu is bigger than that of 5 x 103 pfu. The

QCM

Microfluidic

channel

Oscillation

circuit Connector

Fig 2: (a) Photograph of the fabricated chip. QCM is fixed directly on the circuit substance.

(b) Schematic diagram of the microfluidic chip. The microfluidic channel was made of PDMS and combined with

QCM directly.

10 mm

8.5 mm

PDMS

QCM

14.8 mm

Microfluidic channel

Thickness : 0.1 mm

Height of channel : 200 μm

Φ3 mm

(a) (b)

Fig 3: (a) The schematic diagram of experimental setup. (b) The design of oscillation circuit.

QCM

Power Supply

Noise Filter

High-pass and

Low-pass Filter

(b) (a) Vcc

Frequency

Counter

Oscillation

Circuit

Power

Supply

Sample

injection

QCM

Wiring

Channel

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difference in frequency change was obvious with the sample concentration change. And from the results of

ffigure 5, the graph of condition 5 shows more significant frequency drop than condition 4.

CONCLUSION

From the results, we can conclude it is possible to detect viral RNA by increasing sensing mass. It is said

that the detection limit of rapid diagnosis kit usually used is about 103~104 pfu. And our chip can detect virus

subtype because of PNA. So by using gold nanoparticles, we can detect virus in detail. Also we confirmed the

possibility of more amplitude by using magnetic nanoparticles than gold nanoparticles. It’ll be possible to

reduce loss of sample, so we might be able to detect virus more rapidly. In this research we proposed the

microfluidic chip to detect viral RNA using QCM. By using QCM chip, viral RNA is detectable by increasing

sensing mass with nanoparticles.

ACKNOWLEDGEMENTS

This work was supported in part by the Management Expenses Grants for National Universities Corpo-

rations from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).

REFERENCES

[1] M. Niimi et al.,, “Virus purification and enrichment by hydroxyapatite chromatography on a chip,”

Sensors and Actuators B 201, (2014), 185-190

[2] T. Liu et al., “Particle Size Effect Of the DNA Sensor Amplified with Gold Nanoparticles”

Langmuir 18, (2002), 5624-5626

[3] M. Rodahl et al., “Frequency and dissipation-factorresponses to localized liquid deposits on a QCM

electrode,” Sensors and Actuators B 37 (1996), 111-116

CONTACT

* T. Masuda ; phone : +81-52-789-5026; masuda@mech.nagoya-u .ac.jp

-80

-60

-40

-20

0

20

0 2 4 6 8 10 12

Condition 1 Condition 4 Condition 5

Time [min] Fig 5: Comparing experimental results of using gold nanoparticles and magnetic nanoparticles.

Amount of viral

RNA [pfu] Nanoparticle

Condition 1 104 -

Condition 4 104 Gold nanoparticles

Condition 5 - Magnetic nanoparticles

Amount of viral

RNA [pfu] Nanoparticle

Condition 1 104 -

Condition 2 - Gold nanoparticles

Condition 3 5 x 103 Gold nanoparticles

Condition 4 104 Gold nanoparticles

-20

-15

-10

-5

0

5

0 2 4 6 8 10 12

Time [min]

Condition 1

Condition 3 Condition 4

Condition 2

Fig 4: Experimental results of condition 1 to condition 4.

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