EP4292714A1 - Vorrichtung zur schnellen pcr-analyse - Google Patents
Vorrichtung zur schnellen pcr-analyse Download PDFInfo
- Publication number
- EP4292714A1 EP4292714A1 EP22179190.8A EP22179190A EP4292714A1 EP 4292714 A1 EP4292714 A1 EP 4292714A1 EP 22179190 A EP22179190 A EP 22179190A EP 4292714 A1 EP4292714 A1 EP 4292714A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- thermocyclic
- plate
- temperature
- graphite
- power supply
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004458 analytical method Methods 0.000 title description 4
- 229910002804 graphite Inorganic materials 0.000 claims abstract description 16
- 239000010439 graphite Substances 0.000 claims abstract description 16
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000001816 cooling Methods 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 238000010438 heat treatment Methods 0.000 claims abstract description 12
- 238000003752 polymerase chain reaction Methods 0.000 claims abstract description 11
- 230000003321 amplification Effects 0.000 claims abstract description 10
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 10
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 5
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 5
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 5
- 239000007795 chemical reaction product Substances 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 24
- 230000005284 excitation Effects 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 230000003595 spectral effect Effects 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 2
- 239000000835 fiber Substances 0.000 claims description 2
- 230000002687 intercalation Effects 0.000 claims description 2
- 238000009830 intercalation Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims 2
- 101000909122 Homo sapiens Cytochrome P450 4F2 Proteins 0.000 claims 1
- 102000054767 gene variant Human genes 0.000 claims 1
- 238000012544 monitoring process Methods 0.000 claims 1
- 239000000047 product Substances 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 7
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 7
- 229910052782 aluminium Inorganic materials 0.000 description 7
- 239000000243 solution Substances 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 239000000975 dye Substances 0.000 description 4
- 239000002826 coolant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000017525 heat dissipation Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 230000005672 electromagnetic field Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L9/00—Supporting devices; Holding devices
- B01L9/06—Test-tube stands; Test-tube holders
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0654—Lenses; Optical fibres
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
Definitions
- the present invention relates to a PCR apparatus for nucleic acid amplification.
- thermocyclic plate (2) made of material that allows the temperature to increase and drop as fast as possible. In the analysis of various temperature characteristics of materials suitable for ultra-fast temperature change, a graphite was selected - a material with high thermal conductivity and low specific heat capacity.
- thermocyclic plate (2) When comparing graphite with aluminum, it was found that graphite thermocyclic plate (2) of the same shape (equal volume) requires about 40% less energy compared to aluminum thermocyclic plate (2). Graphite allows for higher heating/cooling rates compared to commonly used aluminum.
- the closest prior art document CN203921614U uses a thermocyclic plate (2) comprising thin graphite elements between layers of other materials.
- the apparatus described herein uses a thermocyclic plate (2) made entirely of graphite allowing faster temperature changes.
- Fig. 1 3D representation of the apparatus for nucleic acid amplification.
- the device consists of four main modules:
- the power supply module consists of a high-current battery and an external power supply / charging unit.
- the PCR device can operate on both internal battery power and an external power supply.
- the internal battery is equipped with a protection module that controls the charging / discharging currents, balances the charging of the cells, protects against excessive discharge.
- the external power supply / charging unit can generate up to 22A at 16.8 V.
- the external operation of the power supply is organized in such a way that the batteries are also charged when the device is operated from an external power supply.
- the control module constantly monitors the battery charge level and displays it on the screen. If the charge level is insufficient for one procedure, the control module will not start the procedure until the unit is connected to an external power supply or charged enough to perform at least 1 complete procedure.
- the temperature module consists of a thermocyclic plate (2), a heating / cooling element and an active heat dissipation system.
- a material with high thermal conductivity and low specific heat capacity - graphite - was selected.
- graphite thermocyclic plate (2) of the same shape requires about 40% less energy compared to aluminum thermocyclic plate (2).
- Graphite allows for higher heating / cooling rates compared to commonly used aluminum.
- the plate was provided with 2 wells (5) for 0.2 ml tubes 2 channels for fluorescence excitation and 2 channels for fluorescence collection.
- thermocyclic plate (2) design was optimised to have high surface area to mass ratio.
- a 430W Peltier element was selected.
- the active heat dissipation system consists of an aluminum radiator and an active fan that blows heat away from the radiator.
- the active cooling element is selected to dissipate 450W of the heat generated by cooling the thermocyclic plate (2).
- the most efficient energy transfer conditions of the Peltier element were maintained to ensure the maximum rate of temperature change.
- the energy transfer of the damper element is highest when the temperature difference between the hot and cold sides is smallest.
- the radiator temperature was actively controlled by regulating the fan speed.
- Evaporation of the solution occurs when the sample is heated. Due to the extremely small volume of the sample, all of the solution evaporates and amplification does not occur, although the liquid condenses and drains to the bottom of the tube as it cools. For this purpose, a higher temperature must be maintained at the top of the tube to prevent evaporation. Amplification experiments showed that the temperature difference between the enclosure and the maximum thermocyclic plate (2) should be in the range of 5-10 °C for optimal amplification efficiency.
- the fluorescence excitation-detection module consists of light sources (1), filters (3) and detectors (6).
- the components of a fluorescence excitation detection system are closely related. Excitation should be selected in the spectral range where dye absorption is highest and recording in the spectral range where dye fluorescence is most intense. However, the absorption and fluorescence bands overlap and these areas of maximum intensities are adjacent to each other at a distance of about 20 to 30 nm, so that with conventional color filters, the excitation light enters the detector (6), thus reducing the overall sensitivity of the system.
- a combination of LED and excitation / detection filters (3) was chosen to ensure no overlap in the excitation and detection areas and a high sensitivity for fluorescence detection.
- the last important component in the fluorescence excitation detection module is the detector (6).
- the volume of the test solution is very small (-10 ul) and the fluorescence intensity is very low, so the detector (6) must be extremely sensitive.
- the detector (6) must be fast enough to record fluorescence at a stable temperature point.
- a fast, high-sensitivity, PIN-type photodiode has been selected for this purpose.
- the photovoltaic diode operating mode is selected to reduce the noise generated by the dark current.
- the photodiode To increase the recording efficiency of the photodiode, it was mounted behind a thermocyclic plate (2) and the fluorescence from the tube to the photodiode was "transported" by a fiber optic (4) system that additionally acts as a spatial filter to reduce the effects of reflections (Add a detailed light removal filtration scheme).
- the photodiodes are additionally actively cooled.
- the photodiodes were equipped with FET-type two-stage operational amplifiers, additionally shielding them from external electromagnetic fields using the Faraday cage principle. In the first stage, the current of the photodiode is converted into a voltage, and in the second stage, this voltage is increased.
- the control module consists of 5 parts - heating / cooling element controller, passive coolant temperature controller, temperature feedback controller, fluorescence controller and process controller.
- the requirements for the heating / cooling element controller are - fast and accurate control of high current in both directions of current flow with minimal heat loss. A multitransistor current control model was chosen for this.
- the passive coolant temperature controller controls the active passive coolant temperature controller, thus ensuring the most efficient energy transfer of the Peltier element.
- the purpose of the temperature feedback controller is to periodically record the thermocyclic plate (2) temperature at high speed and high accuracy and transmit the information to the process controller.
- the process controller has an Integrated ADC converter that digitizes the incoming signal and transmits it for further processing.
- the fluorescence controller consists of two modules - the excitation source controller and the detection module controller.
- the excitation source controller must be stabilized to ensure uniform excitation intensity as operating conditions change.
- the controller of the detection module must ensure the amplification of the weak fluorescence signal and efficient noise filtering.
- One of the most complex components of a device is the process controller. It must be able to receive information from different input devices, process digital and analog signals, control different types of controllers, and present the processed information to output devices.
- a program and a set of algorithms were written for parameter registration, analysis and process control. Fuzzy logic algorithm was used to control the temperature of the thermocyclic plate (2). The algorithm compares the measured actual thermocyclic plate (2) temperature with the set one, which depends on the selected test type. The difference between the measured and set temperature is multiplied by a fixed constant and converted to time. This time is used to generate a modulation control signal (PWM) for the Peltier module controller.
- PWM modulation control signal
- the temperature in the thermocycler is controlled by recording the temperature of the thermocyclic plate (2). Meanwhile, the contact of the tube with the thermocyclic plate (2) and a certain coefficient of temperature transfer to the solution results in temperature differences between the thermocyclic plate (2) and the solution.
- the temperature in the tube is - 2-5 degrees different from the temperature of the thermocyclic plate (2) in the apparatus. After about 2-3 seconds, the temperature in the tube reaches the set value. Taking into account these temperature differences, the temperature control algorithm was adjusted - the temperature of the plate is raised to such values (experimental and modeling data) that the solution in the cuvette reaches the temperature specified in the procedure.
- the instrument is turned on and the test type is selected (procedure type with pre-recorded specific number of cycles, temperature settings, fluorescence recording settings).
- One type of study is manual, i.e. it is possible to fully select all parameters according to the user's needs.
- the battery charge is estimated and in the event of an insufficient charge level, the selected procedure is notified to the user, the procedure is not allowed to run on battery power only, and the procedure is suggested to be performed when connected to an external power source. If the initial conditions are met, the procedure is initiated.
- the instrument asks you to add a sample. After inserting the sample, the instrument checks that the tube is inserted and after closing the lid, asks you to confirm with the button on the display that we are starting the amplification procedure.
- the hood is heated to maximum capacity.
- the heating of the main thermoblock is switched on.
- both the hood and the main thermoblock reach the set temperatures, a cyclic process is started, the parameters of which are pre-programmed.
- the instrument control is programmed so that the time and temperature parameters of the cycle can be changed within the ranges specified in the instrument characteristics, and fluorescence recording is possible at any stage of the cycle. This provides opportunities to perform unique experiments in the study of the mechanisms of dye intercalation into a DNA molecule.
- the display always shows the number of cycles and, depending on the settings, a curve of the fluorescence intensity and the fluorescence value of the current cycle.
- the data is written to the memory chip in real time. After the procedure (all cycles have been completed), the temperature maintenance devices are switched off. The screen informs you of the end of the procedure, analyzes the data, and displays the result - the answer to the clinical question / task. If necessary, the next procedure can be started immediately by selecting the start new procedure button on the device screen. When this option is selected, you are again asked to select the type of procedure you want to perform and the sequence of steps described above is initiated.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP22179190.8A EP4292714A1 (de) | 2022-06-15 | 2022-06-15 | Vorrichtung zur schnellen pcr-analyse |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP22179190.8A EP4292714A1 (de) | 2022-06-15 | 2022-06-15 | Vorrichtung zur schnellen pcr-analyse |
Publications (1)
Publication Number | Publication Date |
---|---|
EP4292714A1 true EP4292714A1 (de) | 2023-12-20 |
Family
ID=82067432
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP22179190.8A Pending EP4292714A1 (de) | 2022-06-15 | 2022-06-15 | Vorrichtung zur schnellen pcr-analyse |
Country Status (1)
Country | Link |
---|---|
EP (1) | EP4292714A1 (de) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1710017A1 (de) * | 2005-04-04 | 2006-10-11 | Roche Diagnostics GmbH | Thermocyclierung eines mehrere Proben enthaltenden Blockes |
US20080182301A1 (en) * | 2006-03-24 | 2008-07-31 | Kalyan Handique | Microfluidic system for amplifying and detecting polynucleotides in parallel |
WO2010115160A2 (en) * | 2009-04-03 | 2010-10-07 | Helixis, Inc. | Devices and methods for heating biological samples |
CN203921614U (zh) | 2014-06-13 | 2014-11-05 | 杭州博日科技有限公司 | 一种pcr反应容器安放装置 |
-
2022
- 2022-06-15 EP EP22179190.8A patent/EP4292714A1/de active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1710017A1 (de) * | 2005-04-04 | 2006-10-11 | Roche Diagnostics GmbH | Thermocyclierung eines mehrere Proben enthaltenden Blockes |
US20080182301A1 (en) * | 2006-03-24 | 2008-07-31 | Kalyan Handique | Microfluidic system for amplifying and detecting polynucleotides in parallel |
WO2010115160A2 (en) * | 2009-04-03 | 2010-10-07 | Helixis, Inc. | Devices and methods for heating biological samples |
CN203921614U (zh) | 2014-06-13 | 2014-11-05 | 杭州博日科技有限公司 | 一种pcr反应容器安放装置 |
Non-Patent Citations (1)
Title |
---|
KIKUTA Y ET AL: "Expression and molecular cloning of human liver leukotriene B4 omega-hydroxylase (CYP4F2) gene", DNA AND CELL BIOLOGY, MARY ANN LIEBERT, NEW YORK, NY, US, vol. 18, no. 9, 1 September 1999 (1999-09-01), pages 723 - 730, XP002223185, ISSN: 1044-5498, DOI: 10.1089/104454999315006 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11084039B2 (en) | Microfluidic analysis system | |
US20020191826A1 (en) | Polymerase chain reaction system | |
US6940598B2 (en) | Multi-channel optical detection system | |
US7186989B2 (en) | Low thermal mass fluorometer | |
KR100840949B1 (ko) | 다중 채널 광학적 검출 시스템 | |
KR101949829B1 (ko) | 열 순환기 | |
US20100075312A1 (en) | Qpcr analysis apparatus | |
EP3426399B1 (de) | Lichtvermittelte polymerasekettenreaktionsamplifikation und produktdetektionssystem sowie verfahren zur verwendung | |
CN109811038B (zh) | 一种底照式核酸等温扩增检测便携仪器 | |
CN103820316A (zh) | 基于旋转式微流控芯片的实时荧光pcr检测系统 | |
KR102133633B1 (ko) | 핵산증폭반응산물을 실시간으로 검출하는 장치 | |
CN104136597A (zh) | 平衡辐射传递的方法和装置 | |
US9821314B2 (en) | Methods, systems, and applications for solar-thermal microfluidic PCR | |
EP4292714A1 (de) | Vorrichtung zur schnellen pcr-analyse | |
TW201339308A (zh) | 核酸序列擴增檢測裝置 | |
CN111560310B (zh) | 一种随机访问式数字核酸检测装置及使用方法 | |
CN214654911U (zh) | 一种核酸检测系统及核酸检测仪器 | |
KR20110137090A (ko) | 광투과형 온도제어장치 및 이를 구비하는 실시간 검출형 중합 효소 연쇄 반응 장치 | |
Farmer et al. | Hand-held thermal-regulating fluorometer | |
Wang et al. | An integrated software solution for real-time PCR analysis based on microfluidic biochip | |
CN214781858U (zh) | 温控系统及基因扩增设备 | |
CN111304051B (zh) | 一种pcr仪及使用方法 | |
Tsai et al. | Development of Non-contact Composite Temperature Sensing (CTS) for photothermal Real-time quantitative PCR Device | |
EP1738829A1 (de) | Polymerase-Kettenreaktionssystem | |
CN111763616A (zh) | 热循环系统 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION HAS BEEN PUBLISHED |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |