A Study on Enhanced Fluorescence Signal-to-noise by using the Stray

Light Shutter for Quantitative PCR Chip

Liang-Chieh Chao

, Chun-Han Chou

, Hsin-Yi Tsai

, Kuo-Cheng Huang

and Dar-Bin Shieh

Taiwan Instrument Research Institute, National Applied Research Laboratories, Hsinchu, Taiwan

Institute of Oral Medicine, School of Dentistry, National Cheng Kung University, Tainan, Taiwan

Keywords: Quantitative Polymerase Chain Reaction (qPCR), Fluorescence Signal-to-noise, Stray Light Shutter.

Abstract: Quantitative polymerase chain reaction (qPCR) is the most important inspection technique for virus,

especially for coronavirus disease, in this year. The qPCR chip and device were planned to develop because

of the characteristics of fast inspection time, high accuracy and small system volume. Therein, the

fluorescence intensity was the important signal in qPCR device, which represented the positive or negative

reaction after the DNA was amplified and bound on the fluorescence dye, but the fluorescence signal was

easily to be affected by the excitation and scattered light. The mini spectrometer was employed to receive the

fluorescence intensity in PCR chip in this study, and the optical simulation was progressed, and the stray light

shutter (SLS) was added to improve the signal-to-noise ratio (SNR) of fluorescence. The analysis results

showed that the SNR of fluorescence can be enhanced from 3.14 to 16.78 by using the SLS with shape of

extend component aperture, which the protruding structure was at the direction away from center. The results

from this manuscript can provide the important reference information to the developer of qPCR chip, whom

can obtain the high SNR fluorescence signal in qPCR inspection process for disease.

1 INTRODUCTION

Quantitative polymerase chain reaction (qPCR also

called real time PCR, RT PCR) is an important

laboratory technique in molecular biology application

based on the PCR technique, which was one of the

most common method in a large amount of disease

detection, such as virus and bacterial inspection (Espy

et al., 2006). The qPCR technique was widely applied

in clinical disease inspection (Kralik et al., 2017),

especially for the coronavirus disease 2019 in this

year. In qPCR process, the standard double strand

DNA would be separated into two single strand DNA

by increasing the reagent to specific temperature,

which the temperature was about 92-95 ℃ depends

on the reagent. In addition, the specific primer DNA

would bind on the single standard DNA with the

annealing of reagent’s temperature to 58-62 ℃, and

the specific primer DNA will extend while the

temperature raised to about 72 ℃. With the repeat

cycle of increased and decreased temperature of

biological sample included DNA, the amount of DNA

can be amplified with 2 to the 30

power in the PCR

process. It looks on the amplification of a targeted

DNA in the regular PCR process, and it collected the

result within the fluorescent signal instead of post

process such as Gel electrophoresis. There are two

type of methods for products detection in real-time

PCR process: (1) non-specific fluorescent dyes

directly intercalated with any double-stranded DNA

and (2) specific DNA probes connected with

oligonucleotides being labelled with fluorescent

reporter (Bustin et al., 2009). When the DNA of

sample included the specific fragment of disease,

qPCR could provide the quantitative fluorescence

information after each thermal cycle. Therefore, the

fluorescence intensity could present positive or

negative reaction to the tested target DNA. Therein,

in consideration of the inspection cost, except the

usage in clinical diagnosis, most of the experiment

would choose SYBR Green for the fluorescence

sample.

In addition, the previous study had proved that the

SYBR Green can also get the precise result as

TaqMan probes can made. The applicability was

demonstrated by measuring the copy number in three

different genetic contexts, which included the

quantification of gene rearrangement, the detection

and quantification in cell lines and cancer biopsies,

Chao, L., Chou, C., Tsai, H., Huang, K. and Shieh, D.

A Study on Enhanced Fluorescence Signal-to-noise by using the Stray Light Shutter for Quantitative PCR Chip.

DOI: 10.5220/0010362500540060

In Proceedings of the 9th International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS 2021), pages 54-60

ISBN: 978-989-758-492-3

and the detection of deletions in dominant optic

atrophy (Ponchel et al., 2003). The results showed

that the presented assay had important clinical

application and can providing accuracy diagnostic

results in short time. Four gerne expression profiles

(A1, A2A, A2B, A3) of adenosine receptors in breast

cancer tissue were analysed by optimized TaqMan

and SYBR Green quantitative RT PCR (Tajadini et

al., 2014). The result showed that the efficiency for

TaqMan and SYBR Green methods in all genes were

calculated mor than 95 %, and the correlations of

mean normalized data of each gene in two methods

were positive and significant. SYBR Green I-based

duplex qPCR was developed for simultaneous

detection of virus (Zheng et al., 2020). Therein, the

classical swine fever virus (CSFV) and porcine

circovirus 3 (PCV3) were simultaneously inspected

in one sample that amplified by their distinct melting

temperatures. The experimental results showed that

the qPCR with fluorescence dye of SYBE Green was

a reliable diagnostic tool to monitor the disease in

clinical field.

In the qPCR system, the accuracy of the result

strongly depends on the correctness, sensitivity and

resolution of the fluorescent signal. In the commercial

PCR System. The most common fluorescent detector

is modulated CMOS Camera or the photodiode. In

modulated CMOS camera, every pixel has 1 amplifier

with it. Therefore, the detecting signal of the tiny

fluorescence variation can be easier observed than

CCD type camera, and also the driving voltage can be

lower at the same time. Based on the advantages

above, CMOS component has been more and more

common use in the Fluorescence lifetime imaging

microscopy (FLIM) techniques. The researcher

(Chen et al., 2015) used the frequency-domain

fluorescence lifetime imaging microscopy (FD-FLIM)

composed with Olympus TIRFM microscope and

CMOS camera capturing the scattered laser and

analysed the intensity and modulation. The result

proved that the calibration in the CMOS camera

observation would be necessary in the imaging

process, which can minimize the effect of bleaching

or background interference. Anitoa’s ultra-low light

CMOS biosensor was used in a handheld, real-time

qPCR system (BioOptics World, 2015). The several

types of pathogen DNA and RNA, including hepatitis

B/C and E coli. The detection limit of four copies per

sample was achieved and over nine orders of

magnitude in dynamic range. The CMOS biosensor

had the enough sensitivity to replace the

photomultiplier tubes and cooled CCDs in

applications medical and scientific instruments.

However, the colour filter should be used on the

photodiode or CMOS camera when multi colour of

fluorescence signal needed to de detect in one sample

for various diseases. Hence, the optical component

which has the characteristics of spectrum separation

becomes an important component in future

application for detection of compound disease in

clinical inspection field. The spectrometer has the

characteristics of wavelength separation, and the

information of detected light intensity at each specific

wavelength can be recorded independently. In the

study, the spectrometer was employed to receive the

fluorescence intensity in qPCR device. In addition,

the package shape of the spectrometer and the effect

the stray light was analyzed, and the stray light shutter

was investigated to prevent the interference of

fluorescence by excitation light and enhance the

fluorescence signal-to-noise ratio. In the future, we

will consider the design and the results that

investigated in this manuscript, and we can get the

more precise results in different concentration of

DNA than previous device.

2 EXPERIMENTAL PRINCIPLE

AND SETUP

The total power of an incident light is equal to the

outer light, and the power can be divided into the

reflected, transmitted, scattered, and absorbed. There

is fluorescent particle in the liquid in the qPCR

sample, and fluorescence is emitted when the

substance or particle is excited and then absorbing the

light or electromagnetic radiation. Generally, the

fluorescence occurs when an electron of molecule

jumps to ground state by emitting the light from the

excited state, and the emission light ceases nearly

immediately when the excitation light stops.

Therefore, the distribution of emission light intensity

of fluorescence particles can be simulated by the

scattering model.

2.1 Scattering Model Principle

If the substance is a particle, the particle would absorb

the light energy and re-emit the light with different

intensity in different directions. Therein, the Rayleigh

scattering theory was employed when the dimension

of particle was smaller than the light wavelength, and

the Mie scattering theory was used when the particle

size was larger than the wavelength of light. However,

there is lots of calculation time in the light intensity

of each light beam. Total integrated scattering (TIS)

A Study on Enhanced Fluorescence Signal-to-noise by using the Stray Light Shutter for Quantitative PCR Chip

is defined as the ratio of the total power generated by

contributions of scattered radiation included the

forward and backward to the incident radiation,

shown as Fig. 1. In the generally TIS situation

(Harvey et al., 2012), the incident beam on the sample

was nearly normal, and the integration was carried

from the small values to almost 90 degrees. When the

light scattered from the specular reflection is small

and if the substance is surface material, the TIS will

be affected by the surface roughness. In addition, the

bidirectional scattering distribution function (BSDF)

was used to describe the scattered light (Pfisterer et

al.). The phenomenon of BSDF is usually split into

the reflected and transmitted light, which is separated

to the BRDF (bidirectional reflectance distribution

function) and BTDF (bidirectional transmittance

distribution function). In general, the vector I is the

incident light, vector R is the specular light, and the

vector S is the scattered light, shown as Fig. 2.

Therein, the specular light presented the mirror

reflected or scattering light. The projection light of

specular light and scattered light on the plane was

defined as β and β

, and the variation was x. When the

scattering phenomenon was caused by an isotropic

random rough surface, and the dimension of rough

structure was relatively smaller than the wavelength

of scattered light. The ABg scattering model can

usually described the required scattering probability

distribution well and was suitable to the polished

optical surfaces. Therefore, the ABg scattering model

was used to fit to the BSDF which is plotted as a

function of





(Won, 2014). The ABg scattering

model was widely used to evaluate scatter results

through ABg parameters, and written as Eqn. (1).

 











(1)

where A must be equal to 0 or larger than 0. B must

be larger than 1E-12, unless g is equal to 0. If g is

equal to 0, then B can be equal to 0. In addition, no

scattering will occur if A is equal to 0. In the

manuscript, the intensity distribution was analyzed by

ABg model, and the fitting curve was similar to the

Lambertian scattering type. The Lambertian

scattering model was usually used to present the

scattering intensity of uniform rough surface. The

probability of the scattered light on the projection

vector β is the same everywhere in the unit circle, and

the BSDF is 1/π.

Figure 1: The schematic of the total integrated scattering.

Figure 2: The schematic of the total integrated scattering.

2.2 Experimental Setup

The FRED optical engineering software was

employed to simulate the fluorescence distribution

that received by the spectrometer in qPCR chip.

Therein, the excitation light was the blue LED with

wavelength of 465 nm and half angle of ±9°, and the

spectrometer was used to receive the fluorescence. In

addition, the spectrometer can detect the light

intensity of 11 wavelengths because of the nano

interference filter deposited on standard CMOS

silicon sensor, which the detection wavelength was

350-1000 nm. The dimension of sensing area was 780

μm × 520 μm, and the diameter of the detection

aperture was 0.9 mm with height of 1 mm. The gap

between the surface of spectrometer and the qPCR

chip was 1 mm. Due to the bottom of the PCR chip

was sealed by the plastic thin film to prevent

outflowing of liquid reagent, which the reflection

ratio of plastic thin film and chamber of qPCR chip

was set at 30% and 80% in the simulation process,

respectively. The schematic of the experimental setup

was shown in Fig. 3. In addition, the reagent was

composed of water and some biological component,

so the setting parameter of refractive index was 1.336

in the simulation process. The setting parameter of

scattering ratio was 0.6, which means the 100% of

incident light and 60% of light will be scattered.

PHOTOPTICS 2021 - 9th International Conference on Photonics, Optics and Laser Technology

Figure 3: The schematic of simulation and experimental

setup.

Figure 4: The Ray trace of (a) specular light and scattered

light and (b) only scattered light.

3 EXPERIMENTAL RESULT

AND DISCUSSION

Initially, the stray light has an evidently effect in the

actual experiment, and the signal variation of the

fluorescence intensity that received by spectrometer

was small. Therefore, the analysis of the scattered

light in the experimental setup and the solution was

presented to obtain a high signal-to-noise ratio results

for the following actual qPCR experiments. Therein,

the specular light in the simulation software was

determined as the noise because it does not excite the

fluorescence substance and directly reflect, shown as

Fig. 4(a). In addition, the specular light was set as

absorption light, and the real signal received by the

spectrometer was the scattered light that generated by

the fluorescence light, which was defined as the

signal and shown as Fig. 4(b). Therefore, the choose

and the shape of the stray light shutter was designed,

and the light intensity of signal and noise was

analyzed in the following sections.

3.1 Effect of Stray Light

In order to investigate whether the stray light shutter

can enhance the SNR or not, the same gap between

the surface of spectrometer and chamber of qPCR

chip was fixed at 1mm. Initially, there was no stray

light shutter at the light path region, shown as Fig. 5

(a). In the analysis progress, the light distribution of

all rays included specular light (defined as noise) and

scattered light was shown in Fig.6 (a). The light

distribution at the corner can be removed when the

specular light was set as be absorbed, which the light

distribution can present that the only scattering light

from the fluorescence substance and shown as Fig.

6(b). The results showed that the specular light was

generated by the incident light on the chamber of

qPCR chip, so the protruding structure was designed

to occlude the specular light from the side wall of

qPCR chip’s chamber. Therein, the protruding

structure was defined as the stray light shutter (SLS)

and the protruding position was designed at the

semicircle away from the center (Fig. 5 (b)) and near

the center (Fig. 5(c)).

With the SLS, the irradiance of signal (scattering

light) and the noise (specular light) were analyzed

under different scattering ratios. The results showed

that signal was linear relationship to the scattering

ratio by using away from center SLS and without

using SLS, but most signal was occluded by using

near center SLS (Fig.7(a)). In addition, the noise

intensity can be evidently decreased by using the

away from center SLS (Fig.7(b)), and the SNR can be

enhanced about 4 -5 times compared to the SNR

without using SLS (Fig.7(c)). On the contrary, the

SNR of fluorescence by using near center SLS was

lower than that without using SLS. Therefore, the

SLS that with protruding structure away from center

was the important component to enhance the SNR of

fluorescence in qPCR process.

A Study on Enhanced Fluorescence Signal-to-noise by using the Stray Light Shutter for Quantitative PCR Chip

Figure 5: The simulation setup of (a) without stray light

shutter (SLS), (b) with SLS away from centre, and (c) with

SLS near the centre.

Figure 6: The light distribution of (a) all rays and (b) only

scattering rays received by sensor.

3.2 Shape Effect of Stray Light Shutter

The signal decreased obviously when the light was

isolated by the designed structure. Hence, four

different shapes of SLS were designed and employed

to analyze the signal, noise and SNR (Fig. 8(a)).

Therein, there was almost no signal when the shape

of SLS was the same to the aperture of spectrometer

and excitation light (Fig. 8(b)). The signal was

slightly higher with protruding structure of

component half aperture shape than component

aperture. There was the highest signal can be obtained

when the structure between the excitation light and

spectrometer was removed and designed only the

protruding structure away from the center. In addition,

there was 60 % of the highest signal by using the SLS

with shape of extend component aperture. In this

shape, the noise also decreased to the half value

compared to the full aperture. Therefore, the highest

SNR can be evaluated and obtained by using the SLS

with shape of the extend component aperture, which

the SNR was 16.78(Fig. 8(c)).

Figure 7: The (a) signal, (b) noise and (c) SNR of fluorescent

under different scattering ratios by without using SLS and

with using away from centre and near centre SLS.

Figure 8: The (a) the design shape of the stray light shutter

(SLS), (b) the irradiance of signal and noise and (c) the SNR

of fluorescence using different shape of SLSs.

PHOTOPTICS 2021 - 9th International Conference on Photonics, Optics and Laser Technology

3.3 Effect of Detection Angle

From the above results, the suitable shape of SLS was

determined. However, several signal would be

blocked by the package of spectrometer because of

the vertical structure with height of 1 mm. Therefore,

the expand angle of the package of spectrometer was

simulated to detect more rays and obtain the better

package shape of spectrometer, shown as Fig. 9(a).

The irradiance of signal and noise that received by

spectrometer with SLS of extend component aperture

shape and without SLS were analyzed, and the results

showed than the higher signal can be obtained with

the increase of expand angle, shown as Fig. 9(b). The

signal was gradually saturated when the expand angle

was set at 30 and 40 degree. In addition, the SNR with

SLS was about 5-6 times compared to the SNR

without SLS, and the SNR with expand angle of 40

degrees can be enhanced 7.3% relative to without

expand angle (Fig. 9(c)). The comparison of the SNR

under different parameters were summarized in Table

1. Therefore, the spectrometer package can be

modified to expand the detection angle and the SLS

can be added in the actual experimental setup to

enhance the SNR of fluorescence.

Figure 9: The (a) chematic of expand angle of spectrometer

package, (b) the irradiance of signal and noise and (c) SNR

of fluorescence without and with SLS under different

expand angles.

Table 1: This comparison of the SNR under different

parameters.

Expand

angle(°)

SLS

0 40

Without SLS 3.14 3.38

With SLS 16.78 18.02

4 CONCLUSIONS

In the results of qPCR technology, the intensity of

fluorescence signal plays an important role because it

indicated the positive or negative reaction of tested

target disease. Hence, the optical simulation results

can provide the referential information for designing

the experimental setup to obtain the higher SNR

fluorescence signal in qPCR chip. From the

simulation results, the 5 times of SNR of fluorescence

can be enhanced by adding a stray light shutter (SLS)

with protruding structure away from centre. In

relative to the original package and without adding

SLS, the SNR of fluorescence can be enhanced from

3.14 to 18.02 by adding SLS and expanding the 40

degrees of spectrometer package. From the above

simulation results, the better fluorescence signal can

be obtained by referring simulation setup and set up

in the actual experiments in the future, which the

higher fluorescence SNR can present the better

performance of qPCR technology.

ACKNOWLEDGEMENTS

This work was supported in part by the Ministry of

Science and Technology, TAIWAN, under Grants

MOST 109–2221–E–492–010–.

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PHOTOPTICS 2021 - 9th International Conference on Photonics, Optics and Laser Technology