Development Research of an ADT-24 Multichannel Real-time Fluorescence PCR Detection System

This study describes a fluorescence detection system for purified ADT-24 nucleic acids and a PCR detection-integrated machine. The application background and structure of the fluorescence scanning system are described. We designed a confocal 4-channel fluorescence detection scanning head with monochromatic LEDs as the excitation light source, a multipixel photon counter (MPPC) as the optical path detection terminal, and a narrow-band filter and dichroic mirror as the main optical components. This paper discusses in detail the principle of the working system, including optical path selection and standards, optical component selection, channel spectral characteristic analysis, detector selection, performance evaluation, optical path simulation, and sensitivity verification. The design and use of this optical system are also summarized. Finally, a 4-fold dilution of mix reagent was used to test the detection effect of the fluorescence scanning head, and a scheme to control “temperature drift” and optimize performance was proposed. According to the verification results and subsequent live debugging, the debugged results achieved the expected design objectives.


Introduction
In recent years, with the rapid development of molecular biology technology, nucleic acid detection has become a common method for identification of pathogenic microorganisms.In particular, nucleic acid detection played a significant role in the detection of COVID-19 during the recent COVID-19 pandemic.Real-time PCR fluorescence detection is the key technology for nucleic acid detection.Therefore, the development and research of fluorescence detection systems, particularly the development of multicolor, multichannel and multitarget detection modules, is crucial for improving the performance of detection instruments and expanding the scope of their application [1].
Based on the previous relevant research in this field, this paper focuses on the ADT-24 nucleic acid purification and PCR detection integrated machine (hereinafter referred to as the integrated machine) fluorescence scanning system, particularly on the core module of the system's 4-channel (FAM, VIC, ROX, CY5) fluorescence scanning head, and examines the optimization of this structure's design.

Application Background
The structure of the integrated fluorescence scanning system is shown in figure 1.The system has a total of 12 thermal cycle modules.Each module contains 2 reaction tube holes for placing PCR tubes.The 12 thermal cycle modules are placed on the serpentine cooling water tank.The water tank contains inlet/outlet cooling water connectors.During the PCR amplification process, cooling water is continuously injected through the cooling water connector to achieve rapid heat exchange.The 4channel fluorescent scanning head is placed under the water tank and can move quickly while driven by the stepper motor drive system to detect the fluorescent signal emitted in the reaction tube by bottom scanning.

Figure 1.
Overall structure of the fluorescence scanning system.

Optical System Design
The optical system is the key component of the fluorescence scanning detection system and is the core of the fluorescence scanning head [2] Optical component selection: The all-in-one machine adopts a 4-color 4-channel (FAM, VIC, ROX, CY5) fluorescence scanning system.According to the overall design requirements of the scanning head and the design indicators and requirements of each channel optical path, the selection of the functional components in the excitation/emission optical path will directly determine the optical performance.The functional components mainly include LEDs, filters, dichroic mirrors, detectors and convex mirrors.table 1 presents the parameters and requirements of the major optical components.Due to the different fluorescence groups of each channel and the existence of "Stokes shift" [3], the maximum excitation light and emission light wavelengths required by each channel during operation are different, and the emission light colors differ, as shown in table 2. According to the different spectral characteristics of each channel, the selected LED excitation light source, excitation filter, emission filter and matching dichroic mirror are also different, and the performance varies greatly between the different channels.Based on the different excitation light wavelengths required by each channel fluorescence group, the LED light source is a monochromatic light source that matches the dye excitation wavelength.[4] Here, it is necessary to pay attention to the intensity of the light source in order to avoid light "photobleaching" caused by excessive brightness; therefore, the brightness is adjusted through LED series-flow-limiting resistance.The customized optical devices, including the LED of each channel, designed according to the spectral characteristics of each channel and the control index requirements of the optical components are described in table 3. Additionally, table 3 also specifies the customized model of each component and its manufacturer as well as other information.To verify the theoretical feasibility of the selected optical devices of each channel, the spectral characteristics of each channel were measured separately using a spectrometer, and the transmission spectral curve of each channel was obtained with the results shown in figure 3.As shown in figure 3, the light through the excitation filter in each channel is near completely reflected by the dichroic mirror, and the transmittance of fluorescence is greater than 50%.Thus, the selected optical devices meet the performance requirements.
Detector: The detector is the Hamamatsu MPPC (multipixel photon counter) S13360 series, which is different from the traditional PD or PMT.Two surface mount models of MPPC are specified in table 3: 3050PE and 3075PE.Because the quantum efficiency and sensitivity of 3050PE meet the detection requirements, and also because it has a superior pixel rate per unit area, the former is recommended.table 4 shows the electrical/optical properties of MPPC.In particular, gain M, dark count N, operating temperature, operating voltage, and temperature coefficient are specified.
The photon sensitivity Sλ of S13360-3050PE can be obtained from the spectral response curve of figure 4. The sensitivity of MPPC is closely related to the fluorescence wavelength, and in the visible light range, the Sλ is above 8x10 4 A/W.

Simulation
To choose optical devices, modeling in Code V, and then in ZEMAX optical system model was performed according to the selected LED light tracing, and analysis and each channel optimization; figure 2 (b) shows a separate ROX channel excitation; in the emission light path trace simulation view, the channel excitation and emission light path calibration are strong, and fluorescence collection efficiency is relatively high.The same effect was achieved using the same method in the optical path simulation with other channels.

Sensitivity Verification
Traditional methods are mainly used to verify the fluorescence collection efficiency and fluorescence excitation capacity of each channel.[5] Fluorescence collection efficiency verification: the product of the fluorescence power Fτ and the sensitivity Sλ of each channel at the minimum resolution is greater than the dark current Id of MPPC, i.e.,   *   > 1.5  , (  ,as shown in equation ( 2)), proving that the system can detect the fluorescence signal when the excitation light intensity is sufficiently high.
K: Instrument constant related to the integrated transmittance and fluorescence collection efficiency of the optical system; φ: Quantum yield is related to the fluorescence wavelength, and the quantum yield varies among the channels; I0: excitation light intensity, excitation light radiation power; ε: Molecular absorption coefficient units are mol/mL/cm; c: Sample concentration the unit is mol/mL; l: Sample light range the unit is cm; Calculation and evaluation of the fluorescence excitation capacity and fluorescence collection efficiency once again proved that each channel could theoretically detect fluorescence signals under the condition of sufficient excitation light and could detect fluorescent molecules with minimum sensitivity.Subsequent experiments proved that the detection of each channel can meet the minimum sensitivity of 100 copies/mL.

Optical Mechanical Design
Optical machinery is the peripheral auxiliary facility of the optical system, whose main role is to fix the protective optical components to assist in the realization of optical performance [6].It focuses on the optical density of the whole optical path, convenient installation and firm fixation.Driven by the stepin motor transmission system, the all-in-one scanning head frequently increases and slows down and moves continuously over a long distance, requiring the optical parts of the scanning head to be highly integrated, small and lightweight.
After multiple optimizations of the scheme, the all-in-one scanning head adopts customized optical parts and successfully realizes the 4-channel small volume and lightweight integrated design.After integration, the center spacing of each channel is 8.6 mm, the maximum optical axis diameter of the main optical components isφ 6 mm, the overall size is 36 ×23 × 18 (mm), the weight is approximately 40 g, and the working distance is 6-8 mm or 14-19 mm.
Structural design is a major and key factor in achieving complete optical properties [7].During the protocol phase, it should adhere to the premise of ensuring the function as simply as possible and reduce the risk of failure caused by the increase in links.The main aspects that require special attention are as follows: A: The principle of pin positioning, hole adjustment and screw tightening for the frame and main body; B: An elastic compression structure should be set between the lens and the fixing machinery to avoid impact rupture of the lens; C: light handling, in particular, the optical path at the led and after the filter, avoid crossing of the excitation light and interference with the fluorescence signal; D: Stay away from the heat source, prevent performance degradation of optical components due to heat release; E: overall blackening and aging treatment, avoid distortion of the optical path due to component deformation; F: Fill the black sealing line of each component after assembly, avoiding the influence of external stray light.

Validation and Promotion
Experimental verification: Using 20 µL of the quadruple nucleic acid amplification solution MIX preparation system, the CT value was 26 or 28 template (cotton plasmid) in 10 µl, and the amplification detection, system reagents and sample preparation list are shown in table 5.After 45 amplification cycles, the "S" fluorescence curve reached a plateau [8].The calibration output of each channel curve is shown in figure 6.The calibration of each channel curve was Δ CT <1.5, the plateau single channel fluorescence value was extremely different <15%, and each channel curve showed high consistency.Cotton plasmids 10 Performance improvement: the fluorescent scanning head due to the snake cooling water storage, PCR amplification, and thermal cycle module rapid lifting temperature can scan the optical device (particularly the MPPC) output, causing "temperature drift" [9], and can easily lead to features such as output curve "tail" and "baseline rise" to weaken the certainty of the positive identification [10].
Through the scanning head, using aluminum substrate instead of the original PCB as circuit carrier, TEC and temperature sensor control the overall temperature control devices as shown in figure 7 (a); by designing the precise circuit, with the scanning head of actual temperature control at 43°, a temperature accuracy of 0.2°C (temperature AD is less than 150) is obtained, so that the output fluorescence AD within 150 is stable, as shown in figure 7

Conclusion
The multichannel fluorescence detection system is designed to ensure that PCR can achieve high accuracy, high stability and high repeatability in multicolor, multitarget and multisample applications.The following conclusions have been reached: (1) In the development of the ADT-24 all-in-one machine, the key core of the 4-channel fluorescence scanning head, optical system theory analysis, design selection, simulation and calculation, can first be derived from the theoretical detection ability to optimize the scanning head, ensure the correct interpretation of the "S" type curve, avoid mistaken identifications, and reduce design cost.
(2) The experimental results show that the design of the all-in-one fluorescence scanning head is reasonable, and the use of the fluorescence detection scanning system also meets the expected requirements.

Figure 2 .
Figure 2. Optical system structure and simulation.Excitation light path: excitation light route is as follows: (1) light LED monochromatic excitation light is generated, followed by (2) flat convex mirror f1 collimation by (3) excitation light filter EXF light into narrower wavelength narrow band excitation light irradiation, followed by transmission to (4) dichroic mirror DM designed to stimulate the light reflection; for fluorescence transmission, the dichroic mirror reflects the excitation light incident to (5) excitation flat convex mirror f2; after the excitation, flat convex mirror convergence focus on the (9) sample, inducing fluorescence of the dye.Emission light path: excitation light induced fluorescent dye fluorescence after (5) excitation of the flat convex mirror f2 collection into parallel fluorescence beam incident to (4) dichroic mirror DM; after the dichroic mirror DM, transmission is incident on (6) emission filter EMF, the parallel fluorescence

Figure 3 .
Figure 3. Optical path transmittance test diagram of each channel.

F τ :
Fluorescence power incident on the detector light window; h: Planck constant, h=6.6*10 -34 J• S; c: Speed of light in vacuum, c=3.0×10 17 nm/s; n: The number of fluorescent molecules per unit area; η: Flat convex mirror fluorescence collection efficiency, related to lens numerical aperture NA; T: Comprehensive transmittance of the optical path; usually equal to 50% λ: Emission fluorescence wavelength; τ: Fluorescence lifetime; Verification of fluorescence emission capability: According to Bill's law, the product of the fluorescence power F and the MPPC sensitivity Sλ is greater than the dark current of the MPPC at the minimum detection resolution.That is,  *   > 1.5   (F,as shown in equation (3)), proves that the fluorescence excitation capacity is sufficiently high and the system can detect fluorescent molecules at the minimum sensitivity. =  *  *  0 * (1 − 10 −c l )

Figure 7 .
Figure 7. Temperature control scheme and fluorescence/temperature stability test.

Table 1 .
Main parameters and performance control of optical components.

Table 3 .
Selection of channel optical path components.

Table 4 .
Optical properties of the MPPC.

Table 5 .
System fluid and sample configuration.