A lateral flow immunoassay chip based on CMOS lens-free imaging, a preparation method, device and use method thereof

Abstract

The application discloses a kind of based on CMOS lens-free imaging lateral flow immunoassay chip and preparation method, device and use method.The chip includes: printable immunochromatography membrane, lens-free image sensor, chip expansion board, first chip electrode, chip handheld area, light transmission flat protection film, sample pad, binding pad and water absorption pad.The device includes lateral flow immunoassay chip and lateral flow immunoassay instrument;Lateral flow immunoassay instrument includes multifunctional shell, LED unit, microprocessor unit and power supply.The device is small in size, high throughput, high sensitivity, realizes integrated device to the instant detection of multiple target detection objects, improves detection efficiency.

Description

Technical Field

[0001] This invention relates to lateral flow immunoassay technology, and more particularly to a lateral flow immunoassay chip based on CMOS lensless imaging, as well as its preparation method, apparatus, and usage method. Background Technology

[0002] The term POCT (Point of Care Testing) was first formally proposed in 1995. It refers to a testing method performed at the sampling site, using portable analytical instruments and reagents to rapidly obtain test results. Rapid and sensitive POCT for analytes in complex samples is crucial for in vitro diagnostics.

[0003] Lateral flow immunoassay is a unique point-of-care testing (POCT) technique with advantages such as low cost, simplicity, and speed, enabling point-of-care detection of target analytes. It is currently widely used in environmental monitoring, medical diagnostics, and food safety. However, colloidal gold lateral flow immunoassay typically cannot quantitatively detect analytes, failing to meet current needs. Furthermore, existing lateral flow immunoassay techniques often have low throughput, detecting only a single target analyte, resulting in low detection efficiency. Moreover, single-analyte-targeting techniques provide limited information about the sample, and in some cases, the results are not accurate or reliable. Therefore, rapid, accurate, and simultaneous detection of multiple target analytes in a sample is becoming increasingly important. High-throughput detection technologies can reduce sample volume, analysis time, and cost. An ideal high-throughput detection system should combine high-performance equipment with low-complexity systems to meet the World Health Organization's POCT standards.

[0004] Lensless imaging technology is a high-throughput microscopic imaging technique that does not require any lenses to directly place or bring the object under test close to the sensor surface for imaging. Due to its small size and low cost, it is suitable for application in POCT technology, providing immediate detection and long-term monitoring for critically ill patients in resource-limited areas. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a lateral flow immunoassay chip based on CMOS lensless imaging, as well as its preparation method, apparatus, and usage.

[0006] The objective of this invention is achieved through the following technical solution: a lateral flow immunoassay chip based on CMOS lensless imaging, comprising: a printable immunochromatographic membrane, a lensless image sensor, a chip expansion board, a first chip electrode, a chip handheld area, a light-transmitting and flat protective film, a sample pad, a conjugate pad, and an absorbent pad; the lensless image sensor has an imaging field of view with a length of not less than 3.76 mm, a width of not less than 2.7 mm, and a height of not less than 300 μm, and a resolution of less than 1.4 μm; the chip handheld area is mounted at the front end of the chip expansion board, and the surface is provided with a lensless image sensor and a first chip electrode; the lensless image sensor and the first chip electrode... The electrode is connected to a plate; a light-transmitting and flat protective film is coated on the lensless image sensor, and a printable immunochromatographic membrane is disposed on the light-transmitting and flat protective film; the printable immunochromatographic membrane is provided with a detection area, a calibration area, and a quality control area; the sample pad overlaps the front end of the printable immunochromatographic membrane, the conjugate pad overlaps the front end of the detection area, and the absorbent pad overlaps the rear end of the printable immunochromatographic membrane; the detection area contains a detection antibody; the calibration area is divided into four parts and contains cyan ink, magenta ink, yellow ink, or black ink respectively; the quality control area contains a second antibody; the conjugate pad contains signal microspheres with immobilized capture antibodies.

[0007] Furthermore, the lensless image sensor includes an imaging area, a sensor circuit board, and image sensor electrodes.

[0008] Furthermore, the light-transmitting and flat protective film has electrode protection grooves.

[0009] Furthermore, the light-transmitting and smooth protective film uses polydimethylsiloxane material, and the thickness of the light-transmitting and smooth protective film is no more than 100 μm.

[0010] Furthermore, the printable immunochromatographic membrane uses polyvinylidene fluoride material, and the width of the printable immunochromatographic membrane is no greater than 600 μm.

[0011] Furthermore, the signal microspheres are gold nanoparticles, carbon nanoparticles, or colored latex microspheres.

[0012] This invention also provides a method for fabricating a lateral flow immunoassay chip based on CMOS lensless imaging, comprising the following steps:

[0013] (1) The chip expansion board is made of hard resin substrate, and then the chip handheld area is installed at the front end of the chip expansion board, and a lensless image sensor and the first chip electrode are set on the surface of the chip expansion board.

[0014] (2) Preparation of a light-transmitting and flat protective film: Polydimethylsiloxane material was prepared on the lensless image sensor by spin coating. The spin coating speed was set to 1000 rpm. After spin coating, the film was placed at room temperature for 24 hours to form a light-transmitting and flat protective film.

[0015] (3) Preparation of printable immunochromatographic membrane: Using a liquid 3D printing instrument, a pattern was printed on the light-transmitting and flat protective film by casting solution consisting of 12wt% PVDF particles, 85wt% triethyl phosphate solution and 3wt% ethanol solution. The membrane was then immersed in 75% triethyl phosphate solution at 20℃ for 10 min to form a nascent membrane. Subsequently, it was immersed in an ice-water gel bath to solidify the membrane. The membrane was then soaked in deionized water for 24 h to completely exchange the residual solvent and dried. Finally, it was soaked in 1wt% Tween 20 solution for 24 h to obtain the printable immunochromatographic membrane.

[0016] (4) The printable immunochromatographic membrane is divided into a front end, a detection area, a calibration area, a quality control area, and a back end. Then, the detection antibody is coated onto the detection area of ​​the printable immunochromatographic membrane using a liquid 3D printing instrument. The membrane is then placed in a 37°C incubator for 60 min to form the detection area. The calibration area is divided into four parts. 0.5 μL of cyan ink, magenta ink, yellow ink, and black ink are printed onto the four parts of the calibration area using a liquid 3D printing instrument. The membrane is then left to stand at room temperature for 12 h to obtain the calibration area. The second antibody is coated onto the quality control area of ​​the printable immunochromatographic membrane using a liquid 3D printing instrument. The membrane is then placed in a 37°C incubator for 60 min to form the quality control area.

[0017] (5) The sample pad is made of glass fiber membrane and overlaps the front end of the printable immunochromatographic membrane.

[0018] Preparation of the conjugate pad: A glass fiber membrane was selected, and 200 μL of signal microspheres immobilized with capture antibodies were sprayed onto the surface of the glass fiber membrane using a liquid 3D printing instrument. The membrane was then dried in a 37°C oven for 12 hours to form a conjugate pad, which was then attached to the front end of the detection area.

[0019] The absorbent pad is made of pure cotton pulp filter paper and overlaps the back end of the printable immunochromatographic membrane.

[0020] The present invention also provides a lateral flow immunoassay device based on CMOS lensless imaging, including a lateral flow immunoassay chip and a lateral flow immunoassay analyzer;

[0021] The lateral flow immunoassay analyzer includes a multifunctional housing, an LED unit, a microprocessor unit, and a power supply;

[0022] The multifunctional housing is fixedly connected to the LED unit, the microprocessor unit, and the power supply; the multifunctional housing includes a chip socket, a spring-loaded blood collection needle, a rubber base, a device grip area, and a display screen; a second chip electrode is provided on the chip socket; the chip socket is used for inserting and removing the lateral flow immunoassay chip; the second chip electrode is used to connect to the first chip electrode;

[0023] The LED unit includes an LED control circuit and an LED; one end of the LED control circuit is connected to a power supply, and the other end is connected to the LED; the LED is located directly above the lateral flow immunoassay chip.

[0024] The microprocessor unit is connected to the second chip electrode, the display screen, and the LED unit, respectively.

[0025] The power supply is connected to the microprocessor unit.

[0026] Furthermore, the volume of the multifunctional outer shell 2 is no greater than 80mm*40mm*36mm.

[0027] The present invention also provides a method of using a lateral flow immunoassay device based on CMOS lensless imaging, comprising the following steps:

[0028] (1) Set the LED light intensity parameters via the display screen;

[0029] (2) Set the exposure time, ISO and white balance parameters via the display screen;

[0030] (3) Use a spring-loaded lancet to collect blood from the fingertip to obtain the blood to be tested;

[0031] (4) Add the blood to be tested to the sample pad in the lateral flow immunoassay chip and wait for no less than 3 minutes;

[0032] (5) Then insert the lateral flow immunoassay chip into the chip socket;

[0033] (6) The signal intensity changes in the detection area of ​​the lateral flow immunoassay chip are recorded using a lensless image sensor;

[0034] (7) By comparing the optical signal detected without adding the sample with the optical signal after adding the sample, the concentration of various target analytes is obtained through the built-in algorithm of the microprocessor unit.

[0035] (8) Remove the lateral flow immunoassay chip and insert a new lateral flow immunoassay chip for the next round of testing.

[0036] The beneficial effects of this invention are as follows: By integrating a CMOS lensless imaging-based lateral flow immunoassay chip, an LED unit, a microprocessor unit, a power supply, and a multifunctional housing, this invention constructs a CMOS lensless imaging-based lateral flow immunoassay device, which has the advantages of high throughput, quantitative detection, and flexible portability. In this embodiment, the device allows the microprocessor unit to control the LED control circuit outputting control signals, enabling adjustment of the LED's light intensity according to specific needs. The lateral flow immunoassay chip and microprocessor unit enable quantitative detection of target analytes. Through this method, lateral flow immunoassay experiments can be successfully completed in this device, obtaining ideal optical signals and achieving quantitative detection of various target analytes. This invention enables high-throughput lateral flow immunoassay quantitative detection experiments, which is of great significance for the research and development of the field of lateral flow immunoassay. Attached Figure Description

[0037] Figure 1 This is a three-dimensional schematic diagram of a lateral flow immunoassay chip based on CMOS lensless imaging.

[0038] Figure 2 This is a front view of a lateral flow immunoassay chip based on CMOS lensless imaging;

[0039] Figure 3 This is a top view of a lateral flow immunoassay chip based on CMOS lensless imaging;

[0040] Figure 4 This is a schematic diagram of the structure of a lateral flow immunoassay chip based on CMOS lensless imaging;

[0041] Figure 5 A top view of a printable immunochromatographic membrane;

[0042] Figure 6 A top view of the printable immunochromatographic membrane, sample pad, conjugate pad, and absorbent pad;

[0043] Figure 7 This is a three-dimensional schematic diagram of the lensless image sensor in chip embodiment 5;

[0044] Figure 8 This is a three-dimensional schematic diagram of the light-transmitting and flat protective film in chip embodiment 6;

[0045] Figure 9 This is a schematic diagram of a lateral flow immunoassay device based on CMOS lensless imaging;

[0046] Figure 10 This is a three-dimensional schematic diagram of a lateral flow immunoassay analyzer;

[0047] Figure 11 This is a front view of a lateral flow immunoassay analyzer;

[0048] Figure 12 This is a left view of a lateral flow immunoassay analyzer;

[0049] Figure 13 A cross-sectional view of a lateral flow immunoassay analyzer;

[0050] In the diagram, 1-lateral flow immunoassay chip, 2-multifunctional housing, 3-LED unit, 4-microprocessor unit, 5-power supply; 6-lateral flow immunoassay analyzer; 101-sample pad; 102-conjugation pad; 103-absorbent pad; 104-calibration area; 105-printable immunochromatographic membrane; 106-detection area; 107-transparent and flat protective film; 108-imaging area; 109-sensor circuit board; 110-image sensor electrode; 111-chip handheld area; 112-chip expansion board; 113-first chip electrode; 115-quality control area; 116-electrode protection groove; 117-lens-less image sensor; 201-chip socket; 202-spring lancet; 203-rubber base; 204-device grip area; 205-display screen; 301-LED control circuit; 302-LED. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0052] Chip Example 1

[0053] like Figures 1-4 As shown, the present invention provides a lateral flow immunoassay chip based on CMOS lensless imaging, comprising: a printable immunochromatographic membrane 105, a lensless image sensor 117, a chip expansion board 112, a first chip electrode 113, a chip handheld area 111, a light-transmitting flat protective film 107, a sample pad 101, a conjugate pad 102, and an absorbent pad 103.

[0054] The chip handheld area 111 facilitates the insertion or removal of the lateral flow immunoassay chip 1 by the user.

[0055] The lensless image sensor 117 has an imaging field of view with a length of not less than 3.76 mm, a width of not less than 2.7 mm, and a height of not less than 300 μm, and a resolution of less than 1.4 μm.

[0056] The chip expansion board 112 has a chip handheld area 111 mounted at the front end, and a lensless image sensor 117 and a chip electrode 113 are provided on its surface. The lensless image sensor 117 is connected to the first chip electrode 113. A light-transmitting and flat protective film 107 is coated on the lensless image sensor 117, and a printable immunochromatographic membrane 105 is provided on the light-transmitting and flat protective film 107.

[0057] like Figure 5 and Figure 6 As shown, the printable immunochromatographic membrane 105 is provided with a detection area 106, a calibration area 104, and a quality control area 115. The sample pad 101 overlaps the front end of the printable immunochromatographic membrane 105, the conjugate pad 102 overlaps the front end of the detection area 106, and the absorbent pad 103 overlaps the rear end of the printable immunochromatographic membrane 105. The detection area 106 contains a detection antibody. The calibration area 104 is divided into four parts and contains cyan ink, magenta ink, yellow ink, or black ink respectively. The quality control area 115 contains a second antibody. The conjugate pad 102 contains signal microspheres with immobilized capture antibodies.

[0058] The detection area 106, calibration area 104, and quality control area 115 are all located directly above the lensless image sensor 117, which facilitates the lensless image sensor 117 in acquiring the color changes of the detection area 106, calibration area 104, and quality control area 115.

[0059] Chip Example 2

[0060] Based on chip embodiment 1, the chip expansion board 112 can use a rigid resin substrate, which is inexpensive, to expand the space of the lensless image sensor and facilitate the design of the flow path required for lateral flow immunoassay.

[0061] Chip Example 3

[0062] Based on chip embodiment 1, the lensless image sensor 117 can increase the imaging field of view by removing the optical lens of the CMOS camera.

[0063] Chip Example 4

[0064] Based on chip embodiment 1, the lensless image sensor 117 uses the OV5647 from Omnivision, a relatively mature CMOS image sensor. There are several alternatives to this type of CMOS image sensor, as long as they meet the requirements of an imaging field of view of at least 3.76 mm in length, 2.7 mm in width, and 300 μm in height, with a resolution less than 1.4 μm.

[0065] Chip Example 5

[0066] like Figure 7 As shown, based on chip embodiment 1, the lensless image sensor 117 includes an imaging region 108, a sensor circuit board 109, and image sensor electrodes 110; the imaging region 108 is located above the sensor circuit board 109, and the image sensor electrodes 110 are located on both sides of the imaging region 108. The imaging region 108 is capable of highly sensitively acquiring optical signals within the upper imaging field of view. The imaging region 108 is used to acquire the image grayscale signal values ​​on the printable immunochromatographic membrane 105.

[0067] Chip Example 6

[0068] like Figure 6 As shown, based on chip embodiment 5, the light-transmitting flat protective film 107 further includes an electrode protection groove 116 for protecting the imaging area 108 and the image sensor electrode 110. The light-transmitting flat protective film 107 is used to level the imaging area 108, the image sensor electrode 110, the sensor circuit board 109, and the chip expansion board 112.

[0069] Chip Example 7

[0070] Based on chip embodiment 1, the light-transmitting flat protective film 107 uses polydimethylsiloxane material, and the thickness of the light-transmitting flat protective film 107 is no greater than 100μm.

[0071] Chip Example 8

[0072] Based on chip embodiment 1, the printable immunochromatographic membrane 105 uses polyvinylidene fluoride material, and the width of the printable immunochromatographic membrane 105 is no greater than 600 μm.

[0073] Chip Example 9

[0074] Based on chip embodiment 1, the signal microspheres are gold nanoparticles, carbon nanoparticles, or colored latex microspheres.

[0075] Preparation method examples

[0076] A method for fabricating a lateral flow immunoassay chip based on CMOS lensless imaging includes the following steps:

[0077] (1) The chip expansion board 112 is made of hard resin substrate, and then the chip handheld area 111 is installed at the front end of the chip expansion board 112, and a lensless image sensor 117 and a chip electrode 113 are set on the surface of the chip expansion board 112.

[0078] (2) Preparation of the light-transmitting and flat protective film 107: Polydimethylsiloxane material is prepared on the lensless image sensor 117 by spin coating. The spin coating speed is set to 1000 rpm. After spin coating, the film is placed at room temperature for 24 hours to form the light-transmitting and flat protective film 107. The polydimethylsiloxane material is a light-transmitting material, and there are many alternatives, as long as the flatness requirement is met.

[0079] (3) Preparation of printable immunochromatographic membrane 105: Using a liquid 3D printing instrument, a casting solution consisting of 12wt% PVDF particles, 85wt% triethyl phosphate solution and 3wt% ethanol solution was used to print a pattern on the light-transmitting and flat protective film 107. The film was then immersed in 75% triethyl phosphate solution at 20°C for 10 min to gel and form a nascent membrane. Subsequently, the film was immersed in an ice-water gel bath to solidify into a membrane. The film was then soaked in deionized water for 24 h to completely exchange the residual solvent and then dried. Finally, the film was soaked in 1wt% Tween 20 solution for 24 h to obtain printable immunochromatographic membrane 105.

[0080] (4) The printable immunochromatographic membrane 105 is sequentially divided into a front end, a detection area, a calibration area, a quality control area, and a back end. Then, a detection antibody is coated onto the detection area of ​​the printable immunochromatographic membrane 105 using a liquid 3D printing instrument. The membrane is then placed in a 37°C incubator for 60 minutes to form the detection area 106. Taking growth stimulation expressed gene 2 (ST2) as the target analyte, the detection antibody used is ST2 monoclonal antibody. The ST2 monoclonal antibody can be diluted to 1.5 mg / mL with 1x PBS solution. Using a liquid 3D printing instrument, 1 μL of the detection antibody is coated onto one detection area of ​​the printable immunochromatographic membrane 105. The membrane is then placed in a 37°C incubator for 60 minutes to obtain the detection area 106. Depending on the target analyte, a monoclonal antibody of the target analyte can be selected as the detection antibody.

[0081] The calibration area was divided into four parts. Using a liquid 3D printing instrument, 0.5 μL of cyan ink, magenta ink, yellow ink and black ink were printed into the four parts of the calibration area respectively. After standing at room temperature for 12 hours, calibration area 104 was obtained.

[0082] The second antibody was coated onto the quality control area of ​​the printable immunochromatographic membrane 105 using a liquid 3D printing instrument, and then placed in a 37°C incubator for 60 minutes to form the quality control area 115. The second antibody used was anti-mouse IgG, which could be diluted to 1 mg / mL with 1x PBS solution.

[0083] The printable immunochromatographic membrane 105 is placed on a light-transmitting, flat protective film 107.

[0084] (5) The sample pad 101 is made of glass fiber membrane and overlaps the front end of the printable immunochromatographic membrane 105. The sample pad 101 is used to collect samples, separate and filter blood cells and impurities in the samples, and perform sample pretreatment. There are many alternatives to such pretreatment materials, as long as they meet the filtration requirements.

[0085] Preparation of the conjugate pad 102: A glass fiber membrane was selected, and 200 μL of signal microspheres immobilized with capture antibodies were sprayed onto the surface of the glass fiber membrane using a liquid 3D printing instrument. The membrane was then dried in a 37°C incubator for 12 hours to form the conjugate pad 102, which was then attached to the front end of the detection area 106. The conjugate pad 102 serves as a carrier for the antibody-labeled signal microspheres to capture the target analyte. Various alternative materials are available to meet the requirement of easy detachment of the signal microspheres.

[0086] The absorbent pad 103 is made of pure cotton pulp filter paper and is attached to the rear end of the printable immunochromatographic membrane 105.

[0087] The liquid 3D printing instrument used in this embodiment is the Sonoplot Micro plotter II inkjet printer. Instruments with similar functions can also be used for this patterning or micro-volume liquid printing application.

[0088] Device Examples

[0089] like Figure 9 As shown, the present invention also provides a lateral flow immunoassay device based on CMOS lensless imaging, including a lateral flow immunoassay chip 1 and a lateral flow immunoassay analyzer 6. Figures 10-13 As shown, the lateral flow immunoassay analyzer includes a multifunctional housing 2, an LED unit 3, a microprocessor unit 4, and a power supply 5.

[0090] The multifunctional housing 2 is fixedly connected to the LED unit 3, the microprocessor unit 4, and the power supply 5 to form a closed space to prevent external interference, thereby making the lateral flow immunoassay detection conditions more stable. The multifunctional housing 2 includes a chip socket 201, a spring-loaded blood collection needle 202, a rubber base 203, a device grip area 204, and a display screen 205; a second chip electrode is provided on the chip socket 201; the chip socket 201 is used to insert and remove the lateral flow immunoassay detection chip 1; the second chip electrode is used to connect to the first chip electrode 113.

[0091] The multifunctional outer shell 2 has a volume of no more than 80mm*40mm*36mm to achieve portability of the device. The spring-loaded lancet 202 is used for blood collection from the user's fingertip; the rubber base 203 is used to reduce external vibration interference during testing; the device grip area 204 is convenient for the user to hold; and the display screen 205 is used to display the test results.

[0092] The LED unit 3 includes an LED control circuit 301 and an LED 302. One end of the LED control circuit 301 is connected to the power supply 5, and the other end is connected to the LED 302. The LED control circuit 301 is used to provide the light source required for lateral flow immunoassay and to adjust the light intensity output by the LED 302. The LED 302 is located directly above the lateral flow immunoassay chip 1, so that the light emitted by the LED 302 can vertically illuminate the detection area 106 in the lateral flow immunoassay chip 1 to achieve the best illumination effect. The light intensity of the LED 302 is represented by a control signal voltage, ranging from 0V to 5V, which is used to meet the requirements of general lateral flow immunoassay.

[0093] The microprocessor unit 4 is connected to the second chip electrode, the display screen 205 and the LED unit 3 respectively.

[0094] The microprocessor unit 4 is connected to the second chip electrode. After the lateral flow immunoassay chip 1 is inserted into the chip socket 201, the first chip electrode 113 and the second chip electrode are connected. Subsequently, the microprocessor unit 4 can adjust the exposure time, sensitivity and white balance parameters of the lens image sensor 117, and at the same time receive the image grayscale signal value collected by the lensless image sensor 117 and perform automated image analysis to obtain the concentration of the target analyte.

[0095] The microprocessor unit 4 is connected to the display screen 205 and can send the obtained concentration of the target analyte to the display screen 205.

[0096] The microprocessor unit 4 is connected to the LED unit 3 and can be used to adjust the control signal of the LED control circuit 301.

[0097] The power supply 5 is connected to the microprocessor unit 4 and provides power to the lateral flow immunoassay device based on CMOS lensless imaging.

[0098] According to the above technical solution, the present invention, through the device described, enables the microprocessor unit 4 to control the LED control circuit 301 to output control signals, thereby adjusting the light intensity of the LED 302 according to specific needs. The lensless image sensor 117 in the lateral flow immunoassay chip 1 acquires the optical signals of the detection area 106, and the built-in algorithm of the microprocessor unit 4 processes and analyzes the optical signals to achieve quantitative detection of various target analytes. The lateral flow immunoassay device based on CMOS lensless imaging provided in this embodiment involves a lateral flow immunoassay chip 1 and a lateral flow immunoassay analyzer 6. The lateral flow immunoassay analyzer 6 includes a multifunctional housing 2, an LED unit 3, a microprocessor unit 4, and a power supply 5. It is small in size, easy to carry, and capable of quantitative detection of various target analytes.

[0099] Usage Method Examples

[0100] This invention also provides an integrated immunoassay method based on CMOS lensless imaging, which includes the following steps:

[0101] (1) Set the light intensity parameters of LED302 through the display screen 205;

[0102] (2) Set the exposure time, ISO and white balance parameters via the display screen 205;

[0103] (3) Use the spring lancet 202 to collect blood from the fingertip to obtain the blood to be tested;

[0104] (4) Drop the blood to be tested onto the sample pad 101 in the lateral flow immunoassay chip and wait for at least 3 minutes;

[0105] (5) Then insert the lateral flow immunoassay chip 1 into the chip socket 201;

[0106] (6) The image grayscale signal value of the detection area 106 of the lateral flow immunoassay chip 1 is recorded by the lensless image sensor 117 and transmitted to the microprocessor unit 4.

[0107] (7) The microprocessor unit 4 has built-in standard curves of the concentration of various target detection objects and the image grayscale signal value of the detection area. After receiving the image grayscale signal value of the blood to be tested from the lensless image sensor 117, it analyzes and obtains the concentration of the target detection object and sends it to the display screen 205.

[0108] Working principle: After blood is dripped onto sample pad 101, it flows through printable immunochromatographic membrane 105, first to conjugation pad 102, and then to detection area 106. If the blood contains the target analyte, it will react with the detection antibody in detection area 106, and detection area 106 will generate a color signal; if the blood does not contain the target analyte, detection area 106 will not change color; the blood then flows to quality control area 115, where a color signal is generated, indicating that the blood has effectively circulated through all areas; finally, the blood reaches absorbent pad 103.

[0109] Before performing integrated immunoassay, users need to establish standard curves for the concentration and grayscale signal values ​​of six target analytes. Taking growth stimulation expressed gene 2 (ST2) as an example, ST2 can be serially diluted using negative serum samples. The ST2 samples of different concentrations are then subjected to integrated immunoassay to obtain standard curves for the ST2 concentration and the corresponding detection area imaging grayscale signal values. As the concentration of ST2 in the sample increases, the imaging grayscale signal value of the corresponding detection area also increases.

[0110] The concentration of ST2 in the samples was obtained by using integrated immunoassay of blood samples from patients with acute heart failure and blood samples from healthy individuals.

[0111] In addition to the above, it should be noted that the terms "one embodiment," "another embodiment," and "embodiment" used in this specification refer to specific features, structures, or characteristics described in connection with that embodiment, which are included in at least one embodiment described in the general description of this application. The appearance of the same expression in multiple places in the specification does not necessarily refer to the same embodiment. Furthermore, when a specific feature, structure, or characteristic is described in connection with any embodiment, the intention is to suggest that implementing such a feature, structure, or characteristic in conjunction with other embodiments also falls within the scope of this invention.

[0112] 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 principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A lateral flow immunoassay chip based on CMOS lensless imaging, characterized in that, include: The device includes a printable immunochromatographic membrane (105), a lensless image sensor (117), a chip expansion board (112), a first chip electrode (113), a chip handheld area (111), a light-transmitting and flat protective film (107), a sample pad (101), a conjugate pad (102), and an absorbent pad (103); the lensless image sensor (117) has an imaging field of view with a length of not less than 3.76 mm, a width of not less than 2.7 mm, and a height of not less than 300 μm, and a resolution of less than 1.4 μm. μm; the chip extension board (112) has a chip handheld area (111) mounted at the front end, and a lensless image sensor (117) and a first chip electrode (113) are provided on its surface. The lensless image sensor (117) and the first chip electrode (113) are connected; a light-transmitting and flat protective film (107) is coated on the lensless image sensor (117), and a printable immunochromatographic membrane (105) is provided on the light-transmitting and flat protective film (107); the printable immunochromatographic membrane (105) is made of polyvinylidene fluoride material, and the width of the printable immunochromatographic membrane (105) is not greater than 600 μm; the printable immunochromatographic membrane ( 105) is provided with a detection area (106), a calibration area (104), and a quality control area (115). The sample pad (101) overlaps the front end of the printable immunochromatographic membrane (105), the conjugate pad (102) overlaps the front end of the detection area (106), and the absorbent pad (103) overlaps the rear end of the printable immunochromatographic membrane (105). The detection area (106) contains a detection antibody. The calibration area (104) is divided into four parts and contains cyan ink, magenta ink, yellow ink, or black ink respectively. The quality control area (115) contains a second antibody. The conjugate pad (102) contains signal microspheres with immobilized capture antibodies. The preparation method of the printable immunochromatographic membrane (105) includes: using a liquid 3D printing instrument to print a pattern on the light-transmitting and flat protective film (107) using a casting solution composed of 12 wt% PVDF particles, 85 wt% triethyl phosphate solution and 3 wt% ethanol solution; immersing the film in a 75% triethyl phosphate solution at 20 ℃ for 10 min to gel and form a nascent membrane; and then immersing the film in an ice-water gel bath to solidify it into a membrane. The membrane was soaked in deionized water for 24 h to completely remove the residual solvent and then air-dried. Subsequently, it was soaked in 1 wt% Tween20 solution for 24 h to obtain a printable immunochromatographic membrane (105).

2. The lateral flow immunoassay chip based on CMOS lensless imaging according to claim 1, characterized in that, The lensless image sensor (117) includes an imaging area (108), a sensor circuit board (109), and image sensor electrodes (110).

3. A lateral flow immunoassay chip based on CMOS lensless imaging according to claim 2, characterized in that, The light-transmitting flat protective film (107) has electrode protection grooves (116).

4. A lateral flow immunoassay chip based on CMOS lensless imaging according to claim 3, characterized in that, The light-transmitting and flat protective film (107) is made of polydimethylsiloxane material, and the thickness of the light-transmitting and flat protective film (107) is not greater than 100 μm.

5. A lateral flow immunoassay chip based on CMOS lensless imaging according to claim 1, characterized in that, The signal microspheres are gold nanoparticles, carbon nanoparticles, or colored latex microspheres.

6. A method for fabricating a lateral flow immunoassay chip based on CMOS lensless imaging as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) The chip expansion board (112) is made of hard resin substrate, and then the chip handheld area (111) is installed at the front end of the chip expansion board (112), and a lensless image sensor (117) and a first chip electrode (113) are set on the surface of the chip expansion board (112). (2) Preparation of a light-transmitting and flat protective film (107): Polydimethylsiloxane material was prepared on the lensless image sensor (117) by spin coating. The spin coating speed was set to 1000 rpm. After spin coating, the film was placed at room temperature for 24 hours to form a light-transmitting and flat protective film (107). (3) Preparation of printable immunochromatographic membrane (105): Using a liquid 3D printing instrument, a pattern was printed on the light-transmitting and flat protective film (107) consisting of 12 wt% PVDF particles, 85 wt% triethyl phosphate solution and 3 wt% ethanol solution. The film was then immersed in 75% triethyl phosphate solution at 20 ℃ for 10 min to form a nascent membrane. Subsequently, it was immersed in an ice-water gel bath to solidify into a membrane. The membrane was soaked in deionized water for 24 h to completely remove the residual solvent and then air-dried. Subsequently, it was soaked in 1 wt% Tween 20 solution for 24 h to obtain a printable immunochromatographic membrane (105). (4) The printable immunochromatographic membrane (105) is divided into a front end, a detection area, a calibration area, a quality control area and a back end in sequence. Then, the detection antibody is coated on the detection area of ​​the printable immunochromatographic membrane (105) by liquid 3D printing instrument and placed in a 37 ℃ constant temperature chamber for 60 min to form the detection area (106). The calibration area is divided into 4 parts. 0.5 μL of cyan ink, magenta ink, yellow ink and black ink are printed in the 4 parts of the calibration area by liquid 3D printing instrument respectively. After standing at room temperature for 12 h, the calibration area (104) is obtained. The second antibody is coated on the quality control area of ​​the printable immunochromatographic membrane (105) by liquid 3D printing instrument and placed in a 37 ℃ constant temperature chamber for 60 min to form the quality control area (115). (5) The sample pad (101) is made of glass fiber membrane and overlaps the front end of the printable immunochromatographic membrane (105); Preparation of the conjugate pad (102): A glass fiber membrane was selected, and 200 μL of signal microspheres with captured antibodies were sprayed onto the surface of the glass fiber membrane using a liquid 3D printing instrument. The membrane was dried in a constant temperature oven at 37 °C for 12 h to form the conjugate pad (102), which was then attached to the front end of the detection area (106). The absorbent pad (103) is made of pure cotton pulp filter paper and is attached to the rear end of the printable immunochromatographic membrane (105).

7. A lateral flow immunoassay device based on CMOS lensless imaging, characterized in that, Includes the lateral flow immunoassay chip and lateral flow immunoassay analyzer as described in any one of claims 1-6; The lateral flow immunoassay analyzer includes a multifunctional housing (2), an LED unit (3), a microprocessor unit (4), and a power supply (5). The multifunctional housing (2) is fixedly connected to the LED unit (3), the microprocessor unit (4), and the power supply (5); the multifunctional housing (2) includes a chip socket (201), a spring-loaded blood collection needle (202), a rubber base (203), a device grip area (204), and a display screen (205); a second chip electrode is provided on the chip socket (201); the chip socket (201) is used to insert and remove the lateral flow immunoassay chip; the second chip electrode is used to connect to the first chip electrode (113); The LED unit (3) includes an LED control circuit (301) and an LED (302); one end of the LED control circuit (301) is connected to the power supply (5), and the other end is connected to the LED (302); the LED (302) is located directly above the lateral flow immunoassay chip (1); The microprocessor unit (4) is connected to the second chip electrode, the display screen (205) and the LED unit (3) respectively; The power supply (5) is connected to the microprocessor unit (4).

8. The lateral flow immunoassay device based on CMOS lensless imaging according to claim 7, characterized in that, The volume of the multifunctional outer shell (2) is no greater than 80mm*40mm*36mm.

9. A method of using the lateral flow immunoassay device based on CMOS lensless imaging as described in claim 8, characterized in that, Includes the following steps: (1) Set the light intensity parameters of LED (302) via the display screen (205); (2) Set the exposure time, ISO and white balance parameters via the display screen (205); (3) Use a spring-loaded lancet (202) to collect blood from the fingertip to obtain the blood to be tested; (4) Drop the blood to be tested onto the sample pad (101) in the lateral flow immunoassay chip and wait for no less than 3 minutes; (5) Then insert the lateral flow immunoassay chip into the chip socket (201); (6) The signal intensity change of the detection area (106) of the lateral flow immunoassay chip is recorded by a lensless image sensor (117); (7) By comparing the optical signal detected without adding the sample with the optical signal after adding the sample, the concentration of various target analytes is obtained through the built-in algorithm of the microprocessor unit (4). (8) Remove the lateral flow immunoassay chip and insert a new lateral flow immunoassay chip for the next round of testing.

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