TWI707042B - Convective polymerase chain reaction apparatus and optical detecting method thereof - Google Patents

Abstract

A convective polymerase chain reaction apparatus includes a tube, a temperature control unit, at least one light source and a sensor. The tube includes a cavity used to contain a reaction solution having a liquid level measured from a bottom of the cavity to a top surface of the reaction solution. The temperature control unit is disposed adjacent to the tube used to control the temperature of the reaction solution. The at least one light source provides a light beam passing through an incident portion of the tube to reach the reaction solution used to excite fluorescence in the reaction solution, wherein the incident portion is located at a height greater than 1/2 the liquid level measure from the bottom of the cavity. The sensor is used to detect the excited fluorescence. The light beam has an incident direction forming a non-straight angle with a long axis of the tube.

Description

Thermal convection polymerase chain reaction device and its optical detection method

This specification discloses a polymerase chain reaction (PCR) device and its detection method. In particular, it relates to a thermal convective polymerase chain reaction (convective Polymerase Chain reaction, cPCR) device and its optical detection method.

Polymerase chain reaction is a molecular biology technique used to amplify specific deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) fragments. A typical polymerase chain reaction device uses the repeated heating and cooling of a thermocycler to perform repeated thermal cycling steps, and gradually conduct thermal conduction into the reaction tube through a metal block to make deoxyribonucleic acid or Ribonucleic acid fragments have three different temperature cycles at different temperatures: denaturation (95℃), adhesion (45℃-65℃) and extension (72℃). However, the traditional multi-level indirect heating method for thermal cycling steps is time-consuming and bulky, which greatly reduces the detection efficiency and its application range.

The thermal convective polymerase chain reaction is a kind of thermal convection principle that directly heats the reaction vessel containing the reaction liquid to generate a temperature gradient of the reaction liquid and cause the phenomenon of thermal convection. The temperature of the reaction liquid can be changed continuously during the convection process, and the nucleic acid can be amplified by looping along with the reaction liquid convection, shortening the time required for temperature change in the traditional method, and having the advantages of simple structure and low cost. The currently used thermal convection polymerase chain reaction device uses a capillary made of plastic or glass as a reaction vessel, and uses a luminescent source placed under the bottom of the test tube to excite the fluorescent reagent in the tube to generate a fluorescent signal. The detector senses the fluorescent signal to monitor the polymerase chain reaction.

However, since the reaction liquid contains a biological sample (for example, whole blood of a living body), its composition is quite complicated. And during the reaction operation, most of the contents of the reaction liquid (such as proteins, blood cells, etc.) will be deposited on the bottom of the capillary tube, causing interference and obstruction in the excitation light path, making it impossible for light to pass through the tube bottom to excite the fluorescence in the tube. Reagents and fluorescent signals cannot be detected by the detector on the side of the tube, which seriously affects the control quality of the polymerase chain reaction.

Therefore, there is a need to provide an advanced thermal convective polymerase chain reaction device and its optical detection method to solve the problems faced by the prior art.

An embodiment in this specification is to provide a thermal convective polymerase chain reaction device. The thermal convection polymerase chain reaction device includes: a tube, a temperature control unit, at least one light source and a sensor. The column includes a cavity for carrying the reaction liquid. The reaction liquid has a liquid height from the liquid level to the bottom of the cavity. The temperature control unit is adjacent to the pipe column for temperature control of the reaction liquid. At least one light source provides light to enter the reaction liquid through the incident part which is greater than half the height of the liquid from the bottom of the pipe column to excite the reaction liquid to emit fluorescence. The sensor is adjacent to the pipe column for sensing fluorescence. Wherein, an incident direction of light forms a non-flat angle with the long axis of the pipe column.

Another embodiment of this specification is to provide an optical detection method for a thermal convective polymerase chain reaction device. The optical detection method includes the following steps: first, a pipe string is provided, and the pipe string includes a cavity for carrying a reaction liquid, and the reaction liquid has a liquid height from the liquid level to the bottom of the cavity. Then, the light is provided to enter the reaction liquid through the incident part which is greater than one-half the height of the liquid from the bottom of the pipe column to excite the reaction liquid to emit fluorescence. Then, a sensor adjacent to the pipe string is used to sense the fluorescence. Wherein, an incident direction of light forms a non-flat angle with the long axis of the pipe column.

Based on the above, the embodiments of this specification disclose a thermal convective polymerase chain reaction device and its optical detection method. By flexibly arranging the relative position of the sensor and the light incident to the thermal convection polymerase chain reaction chamber, the reaction fluorescent signal received by the sensor is maximized, and the conventional technology is used to install the light source in the reaction chamber At the bottom, causing interference and obstruction of the sensor's optical path. At the same time, by adjusting the cavity, the sensor and the incident angle of light, the space application of the thermal convection polymerase chain reaction device can be more efficient, and the purpose of miniaturization can be achieved.

100, 200‧‧‧Thermal convection polymerase chain reaction device

101‧‧‧Pipe string

101a‧‧‧cavity

101b‧‧‧Bottom of cavity

101c‧‧‧Top cover

101d‧‧‧The long axis of the string

101e‧‧‧scale

102‧‧‧Temperature control unit

102a‧‧‧Groove

103、107、108、109‧‧‧Light source

103a, 107a, 108a, 109a‧‧‧light

104、204‧‧‧Sensor

104a, 204a‧‧‧light receiving direction

105‧‧‧Reaction liquid

105a‧‧‧Liquid level

106A, 106B, 106C‧‧‧ incident part

110‧‧‧Filter

301, 302, 303, 304, 304, 305, 306, 307, 308‧‧‧curve

U‧‧‧Liquid height

θ1, θ2, θ3, θ4, θ5‧‧‧Angle

In order to make the above-mentioned embodiments and other purposes, features and advantages of this specification clearer and easier to understand, a few embodiments are specifically cited together with the accompanying drawings and described in detail as follows: Figure 1A is an embodiment according to this specification A perspective view of the partial structure of a thermal convective polymerase chain reaction device shown; Figure 1B is a side view of a partial structure of the thermal convective polymerase chain reaction device shown in Figure 1A; Figure 2 is based on this specification Another embodiment shows a partial structural side view of a thermal convection polymerase chain reaction device.

Figures 3A to 3D show the fluorescence measured by the sensor after the thermal convective polymerase chain reaction of two reaction liquids containing a whole blood sample and pure water using light sources from different angles. strength.

The embodiments disclosed in this specification relate to a thermal convection polymerase chain reaction device and its optical detection method, which can solve the problem of interference and obstruction of the sensor optical path of the conventional thermal convection polymerase chain reaction device, and achieve The purpose of miniaturizing the thermal convection polymerase chain reaction device. In order to make the above objectives, features and advantages of this specification more comprehensible, a few embodiments are described in detail in conjunction with the accompanying drawings.

However, it must be noted that these specific implementation cases and methods are only examples and are not intended to limit the present invention. The present invention can still be implemented using other features, elements, methods and parameters. The embodiments are only used to illustrate the technical features of the present invention, and not to limit the scope of patent application of the present invention. Those with ordinary knowledge in this technical field will be able to make equivalent modifications and changes based on the description in the following specification without departing from the spirit of the present invention. In the different embodiments and drawings, the same elements will be represented by the same element symbols.

Please refer to FIG. 1A and FIG. 1B. FIG. 1A is a perspective view of a partial structure of a thermal convective polymerase chain reaction device 100 according to an embodiment of the present specification. FIG. 1B is a side view of the partial structure of the thermal convection polymerase chain reaction device 100 shown in FIG. 1A. The thermal convection polymerase chain reaction device 100 at least includes: a pipe column 101, a temperature control unit 102, a light source 103 and a sensor 104.

In some embodiments of the present specification, the tube column 101 may be a long-form tube, such as a capillary, which is made of plastic or glass and has light-transmitting properties. Wherein, the pipe string 101 includes a cavity 101 a for carrying the reaction liquid 105. The reaction liquid 105 carried in the cavity 101a has a liquid height U from the liquid surface 105a to the bottom 101b of the cavity 101a.

The temperature control unit 102 is adjacent to the pipe column 101 for temperature control of the reaction liquid 105. In some embodiments of this specification, the temperature control unit 102 is designed to have a heating block capable of accommodating the pipe string 101, and its appearance is a groove 102a. The groove 102a has a shape and size similar to that of the pipe string 101, so that the temperature control unit 102 can be close to the pipe string 101, and can efficiently transfer heat energy and reduce energy loss.

In this embodiment, the temperature control unit 102 includes a metal heating block, which tightly wraps the bottom of the tube column 101 formed by plastic capillary tubes, can transfer heat to the reaction liquid 105 in the plastic capillary tube, and make the tube column 101 The bottom maintains 90 degrees Celsius. The upper cover 101c of the pipe string 101 may be open, and the top of the pipe string 101 can be maintained at 50 degrees Celsius. Therefore, a temperature gradient from hot to cold is formed, so that the deoxyribonucleic acid or ribonucleic acid in the reaction liquid 105 in the tube column 101 is amplified through continuous temperature cycles.

The light source 103 can be a Light Emitting Diode (LED) chip, a halogen lamp, a tritium lamp, a xenon lamp, a laser source or any combination of the above to provide light 103a with a special color light through The column 101 enters the reaction liquid 105 from the incident portion 106A of the cavity bottom 101b of the cavity 101a which is greater than one-half of the liquid height (1/2U) to excite the reaction liquid 105 to emit fluorescence. It should be said that since the reaction liquid 105 will produce different degrees of sediment according to the type of biological specimen, in order to avoid the interference of the sediment at the bottom of the cavity 101b, it is recommended that the incident part 106A of the light source 103 be arranged at the bottom of the cavity 101b is greater than one-half of the liquid height (1/2U), that is, where there are more fluorescent products, to get good detection results.

In an embodiment of the present specification, the light source 103 may be a single-color light-emitting diode die, which is used to provide red light with a wavelength range substantially between 600 nm and 750 nm, and a wavelength range substantially between 600 nm and 750 nm. Green light in the range of 500 nm to 570 nm or blue light in the wavelength range substantially between 420 nm and 500 nm. In another embodiment of this specification, the light source 103 may be a white light emitting diode crystal grain, and a filter 110 is provided between the light source 103 and the tube column 101 to filter out the white light emitted by the light source 103 Part of the wavelength allows light of a specific wavelength to pass through the filter 110 and the column 101 to enter the reaction liquid 105. It is possible to select (replace) different filters 110 to achieve the purpose of providing light 103a of different colors with a single light source 103.

In some embodiments of this specification, the position of the light source 103 relative to the cavity 101a of the column 101 can be adjusted according to the liquid level 105a of the reaction liquid 105. However, in this embodiment, it is assumed that the reaction liquid 105 placed in the cavity 101a of the column 101 has a fixed volume, and the liquid level 105a and the scale 101e have the same height as an example. Therefore, the liquid surface 105a of the reaction liquid 105 and the position of the light source 103 relative to the cavity 101a of the pipe string 101 can be relatively fixed to a specific scale 101e of the pipe string 101.

The sensor 104 is adjacent to the pipe column 101 for detecting and sensing the fluorescent light emitted by the reaction liquid 105 excited by the light 103a. In an embodiment of the present specification, the sensor 104 may include a photodiode, which is used to convert the received fluorescent light intensity into a current or voltage signal. In another embodiment of the present specification, the sensor 104 may include an optical fiber to transmit the received fluorescent signal to a photoelectric conversion device inside or outside the convective polymerase chain reaction device 100, and then perform subsequent signals deal with.

Among them, the sensor 104 is arranged relative to the light source 103 and the pipe column 101. In an embodiment of the present specification, the sensor 104 has a light receiving direction 104a and the incident direction of the light 103a (as shown by the arrow) sandwiched by an angle substantially between 60° and 180°. In addition, the sensor 104 The angle between the light receiving direction 104a of the detector 104 and the long axis 101d of the pipe column 101 is not a straight angle. The angle θ3 formed by the incident direction of the light 103a provided by the light source 103 and the long axis 101d of the pipe column 101 is not a straight angle.

For example, in this embodiment, the angle θ1 formed by the light receiving direction 104a of the sensor 104 and the incident direction of the light 103a is substantially 120°; the light receiving direction 104a of the sensor 104 and the long axis of the pipe column 101 The angle θ2 formed by 101d is substantially 90°; the angle θ3 formed by the incident direction of the light 103a and the long axis 101d of the pipe column 101 is substantially 30°; and the incident direction of the light 103a and the light receiving direction 104a of the sensor 104 And the long axis 101d of the pipe column 101 are located on the same plane.

In some embodiments of this specification, the thermal convection polymerase chain reaction device 100 may further include other light sources, such as light sources 107 and 108, which can provide light 107a and 108a of the same or different wavelengths as the light 103a, respectively. In this embodiment, the light 107a provided by the light source 107 passes through the pipe column 101 and enters the reaction liquid 105 from the incident portion 106B of the cavity bottom 101b of the cavity 101a, which is greater than half the liquid height (1/2U). . The light 108a provided by the light source 108 passes through the column 101 and enters the reaction liquid 105 from the incident portion 106C of the cavity bottom 101b of the cavity 101a, which is greater than half the liquid height (1/2U). It should be noted that, in this embodiment, the incident direction of the light 108a, the light receiving direction 104a of the sensor 104, and the long axis 101d of the pipe column 101 are perpendicular to each other and not on the same plane. In detail, the light receiving direction 104a of the sensor 104 is substantially perpendicular to the incident direction of the light 108a; and the incident direction of the light 108a is substantially perpendicular to the long axis 101d of the column 101.

In an embodiment, the light receiving direction 104a of the sensor 104, the long axis 101d of the pipe column 101, and the incident directions of the light rays 103a and 107a are on the same plane. The angle θ4 formed by the light receiving direction 104a of the sensor 104 and the incident direction of the light ray 107a is substantially 60°; the angle formed by the incident direction of the light 107a and the long axis 101d of the pipe column 101 is substantially 30°.

It should be noted that the relative positions of the sensor 104, the pipe column 101, and the light sources 103, 107, and 108 are not limited thereto. For example, in some other embodiments of this specification, the light receiving direction 104a of the sensor 204 and the long axis 101d of the pipe column 101 may form a non-right angle. Please refer to FIG. 2. FIG. 2 is a partial structural side view of a thermal convection polymerase chain reaction device 200 according to an embodiment of the present specification. Among them, the thermal convection polymerase chain reaction device 200 has a structure similar to that of the thermal convection polymerase chain reaction device 100. The difference is that the thermal convection polymerase chain reaction device 200 includes a light source 109 and the light receiving direction 204a of the sensor 204 is The long axis 101d of the pipe column 101 forms an included angle θ5 of substantially 60°. In this embodiment, the light receiving direction 204a of the sensor 204, the long axis 101d of the column 101, and the incident direction of the light 109a are on the same plane; and the light receiving direction 204a of the sensor 204 and the incident light 109a The direction is vertical.

Subsequently, the light sources 103, 107, 108, and 109 of different angles shown in Figures 1A, 1B, and 2 are used to heat the two reaction liquids 105 containing whole blood samples and pure water. The convective polymerase chain reaction, and the sensor 104 measures its fluorescence intensity to verify the optical detection effect of the thermal convective polymerase chain reaction device 100.

Please refer to Figure 3A to Figure 3D. The curves 301 and 302 depicted in Figure 3A represent the results measured by the sensor 104 after the thermal convective polymerase chain reaction of the two reaction liquids 105 containing whole blood samples and pure water using the light source 103 Fluorescence intensity. The curves 303 and 304 shown in Figure 3B represent the results measured by the sensor 104 after the thermal convective polymerase chain reaction of the two reaction liquids 105 containing whole blood samples and pure water using the light source 107 Fluorescence intensity. The curves 305 and 306 depicted in Figure 3C represent the results measured by the sensor 104 after the thermal convective polymerase chain reaction of the two reaction liquids 105 containing whole blood samples and pure water using the light source 108 Fluorescence intensity. Curves 307 and 308 shown in Figure 3D represent the results measured by the sensor 104 after the thermal convective polymerase chain reaction of the two reaction liquids 105 containing whole blood samples and pure water using the light source 109 Fluorescence intensity.

It can be seen from the detection results of Figure 3A, Figure 3B, Figure 3C and Figure 3D that light sources 103, 107, 108, and 109 with different incident directions are used for samples containing whole blood and pure water. After the two reaction liquids 105 undergo a thermal convective polymerase chain reaction, the sensor 104 measures the fluorescent signal (as shown by the curves 301, 303, 305, and 307) of the reaction liquid 105 containing a whole blood sample and can be compared with the amount The fluorescent signal (as shown by the curves 302, 304, 306, and 308) obtained by measuring the pure water reaction liquid 105 is obviously separated. Therefore, after deducting the background value of the fluorescent signal of pure water, the fluorescent signal of the actual polymerase chain reaction can still be clearly obtained, showing the adjustment of the relative position of the sensor and light incident on the thermal convective polymerase chain reaction chamber It can solve the problem of interference and obstruction of the sensing light path caused by the deposition of the biological sample in the reaction liquid in the conventional technology.

Based on the above, the embodiments of this specification disclose a thermal convective polymerase chain reaction device and its optical detection method. By flexibly arranging the relative position of the sensor and the light incident to the thermal convection polymerase chain reaction chamber, the reaction fluorescent signal received by the sensor is maximized, and the conventional technology is used to install the light source in the reaction chamber At the bottom, causing interference and obstruction of the sensor's optical path. At the same time, by adjusting the cavity, the sensor and the incident angle of light, the space application of the thermal convection polymerase chain reaction device can be more efficient, and the purpose of miniaturization can be achieved.

Although this specification has disclosed the above with preferred embodiments, it is not intended to limit the present invention. Those who have ordinary knowledge in the technical field to which the present invention belongs can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to those defined by the attached patent application scope.

100‧‧‧Thermal convection polymerase chain reaction device

101‧‧‧Pipe string

101a‧‧‧cavity

101b‧‧‧Bottom of cavity

101c‧‧‧Top cover

101d‧‧‧The long axis of the string

101e‧‧‧scale

102‧‧‧Temperature control unit

102a‧‧‧Groove

103、107、108‧‧‧Light source

103a, 107a, 108a‧‧‧light

104‧‧‧Sensor

104a‧‧‧Light receiving direction

105‧‧‧Reaction liquid

105a‧‧‧Liquid level

106A, 106B, 106C‧‧‧ incident part

110‧‧‧Filter

U‧‧‧Liquid height

θ1, θ2, θ3, θ4‧‧‧angle

Claims (10)

A thermal convective polymerase chain reaction (convective Polymerase Chain reaction, cPCR) device includes: a tube (tube), including a cavity for carrying a reaction liquid, the reaction liquid has a liquid surface to the cavity A liquid height at a bottom; a temperature control unit adjacent to the pipe column for temperature control of the reaction liquid; at least one light source, which provides a light, passes through the pipe column by more than half from the bottom An incident portion of the height of the liquid enters the reaction liquid to excite the reaction liquid to emit a fluorescent light; and a sensor adjacent to the pipe column for sensing the fluorescent light; wherein, a portion of the light The incident direction forms an included angle with a long axis of the pipe string, and the included angle is not a straight angle. The thermal convective polymerase chain reaction device described in item 1 of the scope of patent application further includes a filter, which is arranged between the at least one light source and the tube column to filter out part of the wavelength of the light and allow a specific wavelength The light passes through the incident part and enters the reaction liquid. The thermal convective polymerase chain reaction device described in claim 1, wherein the sensor has a light-receiving direction and the incident direction of the light at an angle substantially between 60° and 180°. The thermal convection polymerase chain reaction device described in item 3 of the scope of patent application, wherein the long axis of the pipe column, the light receiving direction and the incident direction of the light are on the same plane. The thermal convection polymerase chain reaction device described in item 3 of the scope of patent application, wherein the long axis of the pipe column, the light receiving direction and the incident direction of the light are not coplanar. The thermal convection polymerase chain reaction device described in item 5 of the scope of patent application, wherein the long axis of the pipe column, the light receiving direction and the incident direction of the light are perpendicular to each other. The thermal convection polymerase chain reaction device described in item 3 of the scope of patent application, wherein the angle between the long axis of the pipe column and the light receiving direction is not a straight angle. The thermal convection polymerase chain reaction device described in item 7 of the scope of patent application, wherein the light receiving direction is perpendicular to the incident direction of the light. An optical detection method for a thermal convective polymerase chain reaction device includes: providing a pipe column, including a cavity for carrying a reaction liquid, the reaction liquid having a liquid level from a liquid level to a bottom of the cavity Liquid height; providing a light, passing through an incident portion of the column greater than one-half the height of the liquid from the bottom, and entering the reaction liquid to excite the reaction liquid to emit a fluorescence; and providing a sensor , Adjacent to the pipe string for receiving the fluorescent light; wherein, an incident direction of the light and a long axis of the pipe string form an included angle, and the included angle is not a straight angle. The optical detection method of the thermal convective polymerase chain reaction device described in the scope of patent application, wherein the sensor has a light receiving direction and the incident direction of the light is sandwiched between 60° and 180°. One angle.

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