CN115747047B - Photoelectric detection equipment based on ultrahigh frequency acoustic fluid nucleic acid - Google Patents

Abstract

The invention provides ultrahigh frequency acoustic fluid-based nucleic acid photoelectric detection equipment, which comprises a nucleic acid extraction device and a nucleic acid amplification and detection device, wherein the nucleic acid extraction device and the nucleic acid amplification and detection device are connected through a connecting pipe, the nucleic acid extraction device comprises a filter chip, an acoustic fluid chip and a chip substrate which are sequentially arranged, the filter chip and the acoustic fluid chip are fixedly connected with the chip substrate, the filter chip is connected with the connecting pipe, the nucleic acid amplification and detection device comprises a multifunctional chip, a sample bearing chip, an upper computer, an LED light source and a temperature control device, the sample bearing chip is connected with the connecting pipe, the sample bearing chip and the multifunctional chip are sequentially arranged on a fixing frame from top to bottom, the LED light source is arranged at the top of the sample bearing chip, the temperature control device is arranged at the bottom of the multifunctional chip, and the LED light source and the temperature control device are both connected with the upper computer. The invention adopts highly integrated equipment to realize various amplifications, improves the temperature change rate and realizes rapid and efficient amplifications.

Description

A photoelectric nucleic acid detection device based on ultra-high frequency acoustic fluid

Technical Field

The present invention relates to the technical field of nucleic acid detection, and in particular to a nucleic acid photoelectric detection device based on ultra-high frequency acoustic fluid.

Background Art

Polymerase Chain Reaction (PCR) tests are considered the gold standard and most sensitive method for nucleic acid testing; however, to date, they require trained personnel to conduct on-site sampling and transport to the laboratory, where technicians complete the operation in a sterile environment. The equipment is bulky and relatively slow (4-6 hours), and it is impossible to complete integrated rapid testing from sample to result. In addition, whether it is collective collection by centralized personnel or individual collection under regional management, cross-contamination during the collection process cannot be avoided, which increases the risk of detection and limits the use scenarios. Highly sensitive and selective reverse transcription PCR (RT-PCR) technology requires expensive laboratory equipment and technical experience. It is also worth noting that the cumbersome nucleic acid extraction step is used before PCR amplification, which also limits its application in this field.

In the absence of centralized laboratory facilities, it is called Point of Care Testing (POCT). The integration of POCT has become a research hotspot in the field of nucleic acid testing, and several mainstream solutions have also emerged. The first is the direct amplification method, in which a specific lysis reagent is used. After lysis, no extraction or purification is performed, and amplification is performed directly. This method undoubtedly simplifies the operation and speeds up the detection, but the specific lysis reagent increases the detection cost and limits the scope of detection. This type of equipment can only detect a specific one or a few nucleic acid samples. The second is the on-chip magnetic bead method, which integrates the magnetic bead method for extracting nucleic acids into a microfluidic chip and adapts it for POCT detection. This adaptation retains the universality of the magnetic bead method for extracting nucleic acids. After extraction, it can be freely combined with subsequent temperature-varying, isothermal and other detection methods, and has a wide range of applications. However, the magnetic bead method itself is complex and time-consuming. Whether it is manual, pneumatic or electric drive, it will increase the manufacturing cost or the threshold for use. In addition, the above two methods mainly target the difficulties of extraction, and so far, no one has delivered an integrated, integrated detection solution.

There are two main detection schemes: optical detection and electrical detection. Optical detection is further divided into naked eye observation and observation through optical instruments. Naked eye observation is the most convenient detection method, but naked eye observation must introduce some kind of color developing reagent, which increases the detection cost on the one hand, and personal subjective observation will also reduce accuracy and sensitivity on the other hand. Electrical observation requires the introduction of reagents that couple nucleic acid amplification signals to electronic changes on sensors, which has a relatively high research threshold and a narrower application range. The most mainstream optical instrument methods today are still large-scale microscopes or commercial CCD or CMOS cameras. So far, no one has delivered an integrated optical detection solution.

Summary of the invention

In response to the technical problems that existing detection devices have bulky structures and low detection accuracy, the present invention proposes a photoelectric nucleic acid detection device based on ultra-high frequency acoustic fluid, which realizes fast, convenient and efficient nucleic acid extraction and amplification, and effectively improves the accuracy of detection.

In order to achieve the above-mentioned purpose, the technical scheme of the present invention is implemented as follows: a nucleic acid photoelectric detection device based on ultra-high frequency acoustic fluid, including a nucleic acid extraction device and a nucleic acid amplification and detection device, the nucleic acid extraction device and the nucleic acid amplification and detection device are connected through a connecting tube, the nucleic acid extraction device includes a filter chip, an acoustic fluid chip and a chip substrate arranged in sequence, the filter chip and the acoustic fluid chip are fixedly connected to the chip substrate, the filter chip is connected to the connecting tube, the nucleic acid amplification and detection device includes a multifunctional chip, a sample-carrying chip, a host computer, an LED light source and a temperature control device, the sample-carrying chip is connected to the connecting tube, the sample-carrying chip and the multifunctional chip are arranged on a fixed frame from top to bottom in sequence, the LED light source is arranged on the top of the sample-carrying chip, the temperature control device is arranged at the bottom of the multifunctional chip, and the LED light source and the temperature control device are both connected to the host computer.

The filter chip includes an inlet, a filter microcolumn array, a flow channel and an outlet. The inlet is connected to the filter microcolumn array, the outlet is connected to the filter microcolumn array through the flow channel, the outlet is connected to the sample carrying chip through a connecting tube, and the bottom surface of the filter chip is fixedly connected to the chip substrate.

The distance between micro-pillars of the filtering micro-pillar array is smaller than the diameter of the microspheres in the nucleic acid extraction reagent.

The acoustic fluid chip is a solid-mounted resonator, which includes a terminal, a piezoelectric material layer and a ground terminal arranged in sequence from top to bottom. The terminal and the ground terminal are both connected to the chip substrate, and the inlet of the filter chip corresponds to the terminal of the acoustic fluid chip up and down.

The chip substrate comprises a substrate terminal, a grounding substrate and a signal connector. The substrate terminal is connected to the terminal of the acoustic fluid chip, the grounding substrate is connected to the grounding terminal of the acoustic fluid chip, and the signal connector is fixedly arranged at the edge of the chip substrate.

The multifunctional chip includes a thin film filter layer, a photoelectric detection and temperature sensing layer, a glass substrate and a functional layer which are arranged in sequence from top to bottom. The thin film filter layer and the photoelectric detection and temperature sensing layer are arranged on the same side of the glass substrate, and the functional layer is arranged on the other side of the glass substrate. The thin film filter layer is a titanium oxide dielectric layer or a silicon oxide dielectric layer, the photoelectric detection and temperature sensing layer is composed of a-Si:H, and the functional layer is a thin film heater formed by vacuum evaporation deposition of aluminum or chromium and patterning it through standard optical lithography and wet etching processes.

The sample carrying chip comprises a fixedly connected substrate and a sample carrying layer, the substrate is a transparent glass sheet, the sample carrying layer is a porous PDMS layer or PMMA layer, and the substrate is connected to the multifunctional chip.

The temperature control device includes a circuit board and a fan, both of which are connected to a host computer. An upper circuit and a lower circuit are respectively arranged on both sides of the circuit board. The upper circuit is connected to a photoelectric detection and temperature sensing layer, and the lower circuit is connected to a functional layer.

The nucleic acid extraction method is as follows: first, the sample, lysis reagent and extraction microspheres are injected into the inlet, and then the acoustic fluidic chip starts to vibrate to mix the liquid at the inlet, and the nucleic acid lysed by the lysis reagent is adsorbed on the extraction microspheres. After a period of sufficient mixing, the acoustic fluidic chip is closed and the liquid is discharged from the outlet. The microspheres adsorbed with nucleic acids cannot pass through the filtration microcolumn array, thereby achieving nucleic acid extraction and filtration.

The nucleic acid amplification method is as follows: adding elution reagent to the microspheres adsorbed with nucleic acid to elute the nucleic acid sample into the liquid, then transferring the liquid to the sample-carrying chip through the connecting tube at the outlet, controlling the temperature through the host computer, and irradiating the liquid with an LED light source to excite fluorescence, collecting optical signals through photoelectric detection through the functional layer, and determining the occurrence of amplification through optical analysis to determine whether a specific sample exists in the liquid.

The extraction equipment and scheme of the present invention only need to use lysis reagents in the process of nucleic acid extraction, which greatly reduces the complexity of extraction and has both extraction efficiency and application scope. The present invention can achieve high-efficiency extraction within a few minutes with only one or two steps of operation, and has basically no restrictions on the type of samples, and can achieve the extraction of various samples.

The amplification and detection equipment and scheme proposed in the present invention can realize amplification and detection simultaneously. The highly integrated equipment may set the temperature control program to realize various types of amplification, not limited to a certain type, and can subsequently integrate cooling equipment such as fans to increase the temperature change rate of both heating and cooling, thereby realizing fast and efficient amplification. The present invention has the characteristics of high sensitivity, can greatly improve the detection limit, realize accurate and sensitive detection, and can limit the detection band to a predetermined specific range by adding a thin film interference filter, greatly reduce background noise, improve the signal-to-noise ratio of signal acquisition, and further improve sensitivity and detection limit. The photoelectric detection site on the device can be changed by the design of a pre-processed mask, and can complete the detection of multiple sites simultaneously or drive the liquid to realize dynamic detection.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a schematic structural diagram of the present invention.

FIG. 2 is a schematic diagram of the external structure of the present invention.

In the figure, 1 is a filter chip, 11 is an inlet, 12 is a filter microcolumn array, 13 is a flow channel, 14 is an outlet, 2 is an acoustic fluid chip, 3 is a chip substrate, 31 is a signal connector, 4 is a multifunctional chip, 41 is a thin film filter layer, 42 is a photoelectric detection and temperature sensing layer, 43 is a functional layer, 5 is a sample carrying chip, 6 is an LED light source, and 7 is a connecting tube.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

As shown in FIG1 , a nucleic acid photoelectric detection device based on ultra-high frequency acoustic fluid includes a nucleic acid extraction device and a nucleic acid amplification and detection device, the nucleic acid extraction device and the nucleic acid amplification and detection device are connected through a connecting tube 7, the nucleic acid extraction device includes a filter chip 1, an acoustic fluid chip 2 and a chip substrate 3 arranged in sequence, the filter chip 1 and the acoustic fluid chip 2 are both fixedly connected to the chip substrate 3, the nucleic acid amplification and detection device includes a multifunctional chip 4, a sample carrier chip 5, a host computer, an LED light source 6 and a temperature control device, the sample carrier chip 5 and the multifunctional chip 4 are arranged on a fixed frame from top to bottom, the LED light source 6 is arranged on the top of the sample carrier chip 5, the temperature control device is at the bottom of the multifunctional chip 4, and the LED light source 6 and the temperature control device are connected to the host computer. The filter chip 1 is mainly used to separate the sample liquid and the extraction microspheres adsorbed with nucleic acid after the nucleic acid is extracted, the acoustic fluid chip 2 is mainly used to make the filter chip 1 vibrate so that the nucleic acid is fully extracted, and the chip substrate 3 is mainly used to fix the filter chip 1 and the acoustic fluid chip 2, and provide signals to the acoustic fluid chip 2 at the same time. The multifunctional chip 4 is mainly used to heat the sample to amplify the nucleic acid in the sample, the sample-carrying chip 5 is mainly used to carry the sample and provide amplification and detection space for the nucleic acid, and the LED light source 6 is mainly used to irradiate the nucleic acid sample in the sample-carrying chip 5 to stimulate the sample fluorescence. As shown in FIG2 , the nucleic acid extraction device and the nucleic acid amplification and detection device are both arranged in a rectangular parallelepiped housing.

Specifically, the filter chip 1 includes an inlet 11, a filter micro-column array 12, a flow channel 13 and an outlet 14. The inlet 11 is connected to the filter micro-column array 12, the outlet 14 is connected to the filter micro-column array 12 through the flow channel 13, and the outlet 14 is connected to the sample-carrying chip 5 through the connecting tube 7. The lower side of the filter chip 1 is fixedly connected to the chip substrate 3. Among them, the inlet 11 is mainly used to inject samples and provide space for extracting nucleic acids, and the filter micro-column array 12 is mainly used to separate liquids and extract microspheres. Therefore, the distance between micro-columns of the filter micro-column array 12 is smaller than the diameter of the microspheres in the nucleic acid extraction reagent, and the sample extracted with nucleic acid will be discharged from the outlet 14 through the flow channel 13. The acoustic fluid chip 2 is a solid-mounted resonator processed using MEMS technology, including a terminal, a piezoelectric material layer and a ground terminal arranged in sequence from top to bottom. The terminal and the ground terminal are both connected to the chip substrate 3. The inlet 11 of the filter chip 1 corresponds to the acoustic fluid chip 2 up and down. The acoustic fluid chip 2 is arranged at the lower side of the inlet 11, and its main function is to fully mix the sample in the inlet 11 by vibration to ensure that the nucleic acid can be extracted smoothly. The chip substrate 3 includes a substrate terminal, a grounding substrate and a signal connector 31. The substrate terminal is connected to the terminal of the acoustic fluid chip 2, the grounding substrate is connected to the grounding terminal of the acoustic fluid chip 2, and the signal connector 31 is fixedly arranged at the edge of the chip substrate 3. The signal connector 31 is connected to a signal generator, and the signal generator controls the vibration and stop of the acoustic fluid chip 2 by transmitting an AC signal.

When performing nucleic acid extraction, the sample, lysis reagent and extraction microspheres are first injected at the inlet 11, and then the acoustic fluid chip 2 starts to vibrate under the control of the chip substrate 3, so that the liquid at the inlet 11 on the upper side of the acoustic fluid chip 2 is evenly mixed, so that the nucleic acid lysed by the lysis reagent is adsorbed on the extraction microspheres. After a period of sufficient mixing, the acoustic fluid chip 2 is closed, and the excess liquid is discharged from the outlet 14, and the microspheres adsorbed with nucleic acids cannot pass through the filtering microcolumn array 12, thereby realizing convenient and universal nucleic acid extraction and filtration.

In the nucleic acid amplification and detection device, the multifunctional chip 4 includes a thin film filter layer 41, a photoelectric detection and temperature sensing layer 42, a glass substrate and a functional layer 43 arranged in sequence from top to bottom. The thin film filter layer 41 is a titanium oxide or silicon oxide dielectric layer, the photoelectric detection and temperature sensing layer 42 is composed of a-Si:H, and the functional layer 43 is a thin film heater formed by vacuum evaporation deposition of aluminum or chromium and patterning it through standard optical lithography and wet etching processes. The thin film filter layer 41 and the photoelectric detection and temperature sensing layer 42 are both arranged on the same side of the glass substrate, and the functional layer 43 is arranged on the other side of the glass substrate. Among them, the multifunctional chip 4 uses MEMS technology to integrate photoelectric detection, heating, temperature measurement, and thin film filtering on a glass substrate of 5×5 cm in size. The thin film filter layer 41 utilizes the principle of thin film interference to deposit a certain thickness of titanium oxide (TiO2) or silicon oxide (SiO2) dielectric layer on the photoelectric detection and temperature sensing layer 42. The thickness of the deposition corresponds to the wavelength range of the light that selectively passes through, and the light outside this range will be reflected, thereby achieving filtering, reducing background noise while improving the signal-to-noise ratio, and thus improving the sensitivity of detection. The photoelectric detection and temperature sensing layer 42 utilizes the temperature and optical properties of hydrogenated amorphous silicon (anhydrogenated amorphous silicon, a-Si:H) to achieve temperature measurement and conversion of optical signals to current. The principle of temperature measurement is that when the temperature changes, the resistance of a-Si:H will change. The correlation between the two is linear within a certain range. Through pre-calibration, the temperature can be calculated by measuring the resistance value. The principle of photoelectric detection is that when photons are irradiated on a-Si:H, a-Si:H will cause electrons to move within a certain range, thereby forming a current. Similarly, there is a corresponding relationship between the two signals under certain conditions, thereby achieving photoelectric detection. The sample-carrying chip 5 includes a fixedly connected substrate and a sample-carrying layer. The substrate is a transparent glass sheet to ensure that the sample will not leak and is light-transmissive. The sample-carrying layer is a porous PDMS or PMMA layer. The sample-carrying layer is mainly used to load the sample. The position of the PDMS layer or PMMA layer is relative to the photoelectric detection and temperature sensing layer 42. The substrate is connected to the multifunctional chip 4. The temperature control device includes a circuit board and a fan. The circuit board and the fan are both connected to the host computer. The upper circuit and the lower circuit are respectively arranged on both sides of the circuit board. The upper circuit is connected to the photoelectric detection and temperature sensing layer 42, and the lower circuit is connected to the functional layer 43. The upper circuit is used to control the photoelectric detection and temperature sensing layer 42 to collect photoelectric signals and temperature signals, and the lower circuit is mainly used to control the functional layer 43 to heat the sample.

The steps for amplifying and detecting nucleic acids are as follows: add elution reagent to the microspheres adsorbed with nucleic acids to elute the nucleic acid sample into the liquid, then transfer the liquid to the sample-carrying chip 5 through the connecting tube 7 at the outlet 14, control the temperature through the host computer, and use the LED light source 6 to irradiate the liquid on the sample-carrying chip 5 to excite fluorescence, use the photoelectric detection and temperature sensing layer 42 to collect the fluorescence signal through photoelectric detection, and judge whether the nucleic acid is amplified by analyzing the changes in the light signal, and then judge whether there is a specific sample in the liquid.

In view of the problems existing in the existing extraction methods, the present invention introduces MEMS technology and acoustofluidic technology, designs an acoustofluidic nucleic acid extraction chip and method, and realizes fast, convenient and efficient extraction. In view of the problems existing in the existing nucleic acid detection methods, we designed and manufactured a-Si:H that integrates heating and photoelectric detection, and realizes integrated nucleic acid detection.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. A nucleic acid photoelectric detection device based on ultra-high frequency acoustofluidics, characterized in that it comprises a nucleic acid extraction device and a nucleic acid amplification and detection device, the nucleic acid extraction device and the nucleic acid amplification and detection device are connected via a connecting tube (7), the nucleic acid extraction device comprises a filter chip (1), an acoustofluidic chip (2) and a chip substrate (3) arranged in sequence, the filter chip (1) and the acoustofluidic chip (2) are both fixedly connected to the chip substrate (3), the filter chip (1) is connected to the connecting tube (7), the nucleic acid amplification and detection device comprises a multifunctional chip (4), a sample carrier chip (5), a host computer, an LED light source (6) and a temperature control device, the sample carrier chip (5) is connected to the connecting tube (7), the sample carrier chip (5) and the multifunctional chip (4) are arranged on a fixed frame from top to bottom, the LED light source (6) is arranged on the top of the sample carrier chip (5), the temperature control device is arranged on the bottom of the multifunctional chip (4), and the LED light source (6) and the temperature control device are both connected to the host computer; The filter chip (1) comprises an inlet (11), a filter micro-column array (12), a flow channel (13) and an outlet (14); the inlet (11) is connected to the filter micro-column array (12); the outlet (14) is connected to the filter micro-column array (12) via the flow channel (13); the outlet (14) is connected to the sample-carrying chip (5) via a connecting tube (7); and the bottom surface of the filter chip (1) is fixedly connected to the chip substrate (3); The distance between micro-columns of the filtering micro-column array (12) is smaller than the diameter of the microspheres in the nucleic acid extraction reagent. 2. The UHF acoustofluidic nucleic acid photoelectric detection device according to claim 1 is characterized in that the acoustofluidic chip (2) is a solid-mounted resonator, which includes a terminal, a piezoelectric material layer and a ground terminal arranged in sequence from top to bottom, and the terminal and the ground terminal are both connected to the chip substrate (3), and the inlet (11) of the filter chip (1) corresponds to the terminal of the acoustofluidic chip (2) up and down. 3. The UHF acoustofluidic nucleic acid photoelectric detection device according to claim 2 is characterized in that the chip substrate (3) comprises a substrate terminal, a grounding substrate and a signal connector (31), the substrate terminal is connected to the terminal of the acoustofluidic chip (2), the grounding substrate is connected to the grounding terminal of the acoustofluidic chip (2), and the signal connector (31) is fixedly arranged on the edge of the chip substrate (3). 4. The UHF acoustic fluid-based nucleic acid photoelectric detection device according to claim 2 or 3 is characterized in that the multifunctional chip (4) comprises a thin film filter layer (41), a photoelectric detection and temperature sensing layer (42), a glass substrate and a functional layer (43) which are arranged in sequence from top to bottom, the thin film filter layer (41) and the photoelectric detection and temperature sensing layer (42) are both arranged on the same side of the glass substrate, and the functional layer (43) is arranged on the other side of the glass substrate, the thin film filter layer (41) is a titanium oxide dielectric layer or a silicon oxide dielectric layer, the photoelectric detection and temperature sensing layer (42) is composed of a-Si:H, and the functional layer (43) is a thin film heater formed by vacuum evaporation deposition of aluminum or chromium and patterning it through standard optical lithography and wet etching processes. 5. The UHF acoustic fluid-based nucleic acid photoelectric detection device according to claim 4 is characterized in that the sample-carrying chip (5) comprises a fixedly connected substrate and a sample-carrying layer, the substrate is a transparent glass sheet, the sample-carrying layer is a porous PDMS layer or PMMA layer, and the substrate is connected to the multifunctional chip (4). 6. According to the ultra-high frequency acoustic fluid based nucleic acid photoelectric detection device according to claim 5, it is characterized in that the temperature control device includes a circuit board and a fan, the circuit board and the fan are both connected to the host computer, and the upper circuit and the lower circuit are respectively arranged on both sides of the circuit board, the upper circuit is connected to the photoelectric detection and temperature sensing layer (42), and the lower circuit is connected to the functional layer (43). 7. The UHF acoustofluidic nucleic acid photoelectric detection device according to claim 6 is characterized in that, when used for nucleic acid extraction, the sample, lysis reagent and extraction microspheres are first injected into the inlet (11), and then the acoustofluidic chip (2) starts to vibrate to mix the liquid at the inlet (11), so that the nucleic acid lysed by the lysis reagent is adsorbed on the extraction microspheres. After a period of sufficient mixing, the acoustofluidic chip (2) is closed and the liquid is discharged from the outlet (14), and the microspheres adsorbed with nucleic acids cannot pass through the filtering microcolumn array (12), thereby achieving nucleic acid extraction and filtration. 8. The UHF acoustic fluid-based nucleic acid photoelectric detection device according to claim 7 is characterized in that, when used for nucleic acid extraction, it also includes adding an elution reagent to the microspheres adsorbed with nucleic acid to elute the nucleic acid sample into the liquid, and then transferring the liquid to the sample-carrying chip (5) through the connecting tube (7) at the outlet (14), controlling the temperature through the host computer, and irradiating the liquid with an LED light source (6) to excite fluorescence, collecting optical signals through photoelectric detection and a temperature sensing layer (42), and determining the occurrence of an amplification phenomenon through optical analysis, thereby determining whether a specific sample exists in the liquid.