Long afterglow fluorescent time-resolved gene chip labeling and reading method and system

Abstract

The application discloses a long-afterglow fluorescent time-resolved gene chip marking and reading method and system. The method comprises the following steps: adding biotinylated nucleotides in a nucleic acid amplification or reverse transcription process, so that target nucleic acid products carry biotin labels; hybridizing the target nucleic acid products with a gene chip probe array; and then using streptavidin coupled with a long-afterglow fluorescent marker to combine with the biotin, so that long-afterglow fluorescent signals are formed at corresponding probe sites. The system comprises a pulsed excitation light source module, an optical module, a planar array fluorescent detection module, a time sequence control module and a data processing module. After the pulsed excitation light source stops outputting excitation light, the time sequence control module opens an acquisition window after a preset delay time, and time-resolved acquisition is performed on the long-afterglow fluorescent signals, so that the interference of short-life background, substrate spontaneous fluorescence and scattered light is reduced. The data processing module obtains target nucleic acid detection results or interpretation results according to probe site signals.

Description

Technical Field

[0001] This invention relates to the fields of gene chip reading, nucleic acid labeling, fluorescence detection, molecular biology detection, and photoelectric imaging technology. Specifically, it relates to a labeling method and reading system for gene chip reading by conjugating biotinylated nucleotides and streptavidin with long-persistent fluorescent markers and using delayed-gated time-resolved acquisition. It is particularly suitable for applications such as high-density targeted resequencing gene chips, detection of preset pathogen targets, detection of variant sites, and detection of drug resistance genes. Background Technology

[0002] Gene chip technology immobilizes a large number of nucleic acid probes on a solid substrate, enabling specific hybridization between target nucleic acid fragments in the sample and the corresponding probes. The hybridization results are then read using fluorescence, chemiluminescence, or other detectable signals. This technology features parallel detection, multi-target integration, high standardization, and clear data structure, making it suitable for applications such as pre-defined pathogen target detection, drug resistance gene locus detection, variant site detection, genotyping marker site detection, and gene expression profiling analysis.

[0003] In high-density gene chips and targeted resequencing chips, a large number of probe sites are typically deployed on the chip surface. For the same target site, multiple candidate probe sites can be set, and the interpretation is based on the signal differences between different probe sites. Therefore, the reading system not only needs to have high detection sensitivity, but also low background noise, a large dynamic range, and good ability to distinguish adjacent sites.

[0004] Traditional gene chip reading systems typically use common organic fluorescent dyes to label target nucleic acids and read the fluorescence signal through continuous illumination, laser scanning, or area array camera imaging. The fluorescence lifetime of common organic fluorescent dyes is usually on the order of nanoseconds, and their target signal is easily overlapped temporally with the autofluorescence of the chip substrate, scattered light, stray light, and short-lived background signals in the reaction system. For low-abundance target nucleic acids, these background signals can reduce the detection signal-to-noise ratio; for high-density probe arrays, strong signal sites may also experience local saturation, halos, or crosstalk between adjacent sites, thus affecting signal quantification and probe set interpretation.

[0005] To improve detection sensitivity, existing gene chip reading devices typically employ confocal scanning optical paths, high-performance scientific cameras, strong excitation light sources, long-term averaging acquisition, or complex low-noise detection structures. While these methods can improve detection performance to some extent, they increase equipment costs, system complexity, and maintenance difficulty, hindering the miniaturization, engineering, and large-scale deployment of gene chip detection equipment.

[0006] Time-resolved fluorescence detection technology utilizes the difference in fluorescence lifetime between the target fluorescence signal and the background signal. By delaying the opening of the acquisition window after the excitation light is turned off, the target signal and the short-lived background signal are separated in the time dimension. In particular, long-afterglow fluorescent labels such as europium ions and terbium ions, which are lanthanide rare earth complexes, can maintain detectable fluorescence signals on the microsecond to millisecond timescale after the excitation light is turned off, making them suitable for delayed-gated acquisition. However, current time-resolved fluorescence technology is more often used in immunoassay, fluorescence analysis, or general imaging fields, and has not yet been fully integrated with the gene chip detection needs such as nucleic acid amplification labeling, gene chip hybridization, area array imaging readout, multi-time window fusion, and high-density probe set interpretation.

[0007] Therefore, there is an urgent need for a long-persistence fluorescence labeling method and a time-resolved reading system suitable for gene chips, which can achieve effective labeling during the preparation of target nucleic acids, form an amplifiable long-persistence fluorescence signal after chip hybridization, and reduce background interference and alleviate saturation and crosstalk problems by delaying acquisition after excitation and fusing multiple time windows, thereby achieving high signal-to-noise ratio and large dynamic range gene chip reading with low hardware complexity.

[0008] Several invention patents have been filed to address the issues of improving fluorescence imaging sensitivity and reducing costs. For example:

[0009] CN110231321A discloses a super-resolution microscopy imaging system based on nanopore-microlens scanning. Its focus is on achieving super-diffraction-limited focusing of the light spot through nanopore-microlens and combining this with scanning structures to achieve fluorescence imaging. This approach primarily focuses on super-resolution scanning imaging structures and does not address the introduction of biotin-labeled nucleotides during nucleic acid amplification or reverse transcription, the use of streptavidin coupled with long-persistent fluorescent markers to form long-persistent fluorescence signals for gene chips, or delayed-gated array readout and multi-time-window fusion interpretation for high-density gene chips.

[0010] CN109297905A discloses a dual-gated correlated fluorescence imaging device and method, which acquires fluorescence time-resolved images at different delays using a narrow-pulse laser, a gated image enhancement charge-coupled device, and a timing control unit. This scheme primarily focuses on the dual-gated correlated fluorescence imaging process and does not address biotinylation labeling of target nucleic acids in gene chip detection, signal amplification of streptavidin-conjugated long-persistence fluorescent labels, low-complexity integral imaging based on area array image sensors, or the interpretation of target nucleic acids under high-density four-probe or multi-probe group structures. Summary of the Invention

[0011] The purpose of this invention is to provide a long-persistence fluorescence time-resolved gene chip labeling and reading method and system. By introducing biotinylated nucleotides during the preparation of target nucleic acids and using streptavidin-conjugated long-persistence fluorescent labels for binding and signal amplification, and by delaying acquisition after excitation, acquiring data in multiple time windows, and interpreting probe arrays, the invention reduces the interference of autofluorescence, short-lived background fluorescence, and scattered light from the chip substrate, improves the detection capability of low-abundance target nucleic acids and the dynamic range of the system, and enhances the signal resolvability between high-density probe sites.

[0012] To achieve the above objectives, the present invention provides a method and system for labeling and reading gene chips with long afterglow fluorescence time-resolved resolution.

[0013] The method includes: adding biotinylated nucleotides during the nucleic acid amplification or reverse transcription reaction of the sample to be tested, and incorporating the biotinylated nucleotides into the newly synthesized nucleic acid chain under the action of polymerase or reverse transcriptase to obtain a target nucleic acid product carrying a biotin label; hybridizing the target nucleic acid product with a probe array on the surface of a gene chip; binding streptavidin coupled with a long-persistent fluorescent label to the biotin label on the target nucleic acid product, thereby forming a long-persistent fluorescent signal at the corresponding probe site; irradiating the gene chip with a pulsed excitation light source, and after the pulsed excitation light source stops outputting excitation light, opening the acquisition window again after a preset delay time to perform time-resolved acquisition of the long-persistent fluorescent signal; performing background correction, intensity extraction or fusion processing on the acquired time-resolved fluorescent signal, and generating at least one of the following based on the time-resolved fluorescent signal intensity of each probe site, probe site coordinates and probe array design information: target nucleic acid detection result and interpretation result.

[0014] The system includes a gene chip, a pulsed excitation light source module, an optical module, an area array fluorescence detection module, a timing control module, and a data processing module. The gene chip is used to fix the probe array; the pulsed excitation light source module provides pulsed excitation light to the gene chip; the optical module guides the excitation light to the gene chip and collects the long-persistence fluorescence signal emitted by the gene chip; the area array fluorescence detection module collects the long-persistence fluorescence signal within a collection window; the timing control module is connected to both the pulsed excitation light source module and the area array fluorescence detection module, and controls the area array fluorescence detection module to open the collection window after a preset delay time after the pulsed excitation light source module stops outputting excitation light; the data processing module generates at least one of the following based on probe site coordinates, time-resolved fluorescence signal, and probe array design information: target nucleic acid detection result and interpretation result.

[0015] The long-persistence fluorescent markers referred to in this invention are fluorescent markers that have a fluorescence lifetime in the microsecond to millisecond range after the excitation light is turned off, and can generate detectable fluorescence signals within a delayed acquisition window. The long-persistence fluorescent markers may include lanthanide rare earth complex fluorescent markers, such as europium ion complexes, terbium ion complexes, or combinations thereof. In one embodiment, the streptavidin can be coupled with a europium ion complex as a long-persistence fluorescent marker, the emission peak of which can be located in the range of 610 nm to 625 nm, and the fluorescence lifetime can be in the range of 100 μs to 2000 μs; in another embodiment, the streptavidin can be coupled with a terbium ion complex as a long-persistence fluorescent marker, the emission peak of which can be located in the range of 535 nm to 550 nm. The specific excitation wavelength, emission filter, and acquisition window can be configured according to the excitation spectrum, emission spectrum, and lifetime parameters of the selected marker.

[0016] The biotinylated nucleotide is a biotin-labeled nucleotide that can be recognized by polymerase or reverse transcriptase and incorporated into a newly synthesized nucleic acid chain, including but not limited to biotin-labeled uracil deoxynucleotide, biotin-labeled cytosine deoxynucleotide, Biotin-11-dUTP, Biotin-11-dCTP, or combinations thereof. The molar ratio of biotinylated nucleotides in the corresponding natural nucleotides can be adjusted according to the target nucleic acid length, amplification efficiency, hybridization efficiency, labeling density, and subsequent fluorescence intensity requirements.

[0017] In one embodiment, to avoid the adverse effects of excessively high biotinylated nucleotide incorporation on the chain conformation, amplification efficiency, and hybridization efficiency with the probe array of the target nucleic acid product, the molar proportion of the biotinylated nucleotide in the corresponding natural nucleotide is no higher than 25%. In a further embodiment, the molar proportion can be from 1% to 25%, and can be optimized according to the amplification system, polymerase or reverse transcriptase type, target nucleic acid length, probe length, hybridization conditions, and required fluorescence signal intensity.

[0018] Streptavidin conjugated with a long-persistent fluorescent label can be added before, during, or after hybridization of the target nucleic acid product with the probe array. In one embodiment, the streptavidin is added after the target nucleic acid product has hybridized with the chip probe array and been washed to reduce the influence of the label on hybridization efficiency and steric hindrance. Since a single target nucleic acid product can carry multiple biotinylate labeling sites, and a single streptavidin molecule can be conjugated with one or more long-persistent fluorescent labels, multi-site binding and signal enhancement can be achieved.

[0019] The reading system can set multiple acquisition windows after the same excitation to acquire long-afterglow fluorescence signals at different decay stages, and then fuse the signals obtained from multiple acquisition windows. The fusion processing may include dark field subtraction, flat field correction, background subtraction, time window normalization, saturation judgment, weak signal integration enhancement, and weighted fusion of images from different time windows. When the signal in the early window reaches or exceeds the saturation threshold, the signal from the middle or late window can be used for replacement or correction; when the signal is below the weak signal threshold, the integrated signal from a longer acquisition window can be used for enhancement.

[0020] When the gene chip is a high-density targeted resequencing gene chip, the probe array may include multiple probe sets, each corresponding to a base position to be interpreted in the target reference sequence. Each probe set includes at least four probe sites, each of which is immobilized with a nucleic acid probe targeting a different candidate base. During data processing, the base type, mutation status, or genotyping result of the corresponding base position to be interpreted can be determined based on the time-resolved fluorescence signal intensity differences at different probe sites within the same probe set. Beneficial effects

[0021] 1. By incorporating biotinylated nucleotides during amplification or reverse transcription and binding them with streptavidin-conjugated long-afterglow fluorescent markers, the target nucleic acid product can carry multiple fluorescent markers, thereby improving the detectability of low-abundance target nucleic acids.

[0022] 2. By delaying the acquisition of long-persistence fluorescence signals after the excitation light is turned off, the time periods of strong spontaneous fluorescence of the chip substrate, short-lifetime background fluorescence, and scattered light can be avoided, thereby improving the detection signal-to-noise ratio.

[0023] 3. By utilizing the microsecond to millisecond-level decay characteristics of long-persistence fluorescence, integrated imaging can be performed using industrial monochrome CMOS cameras, CCD image sensors, or other area array image sensors, which helps to reduce the dependence on high-speed point detectors and complex scanning systems.

[0024] 4. By setting multiple time acquisition windows and fusing them, the impact of strong signal probe site saturation can be reduced, while also taking into account weak signal detection, thereby expanding the dynamic range of the system.

[0025] 5. This invention can be adapted to high-density four-probe or multi-probe step-by-step resequencing chips, and can perform base interpretation or variant identification by the time-resolved fluorescence signal differences of different probe sites within the same probe group. Attached Figure Description

[0026] Figure 1 This is a flowchart of the overall process of the long-persistence fluorescence time-resolved gene chip labeling and reading method and system of the present invention.

[0027] Figure 2This is a schematic diagram illustrating the principle of biotin-labeled nucleotide incorporation and long-afterglow fluorescent labeling in this invention.

[0028] Figure 3 This is a schematic diagram of the time-resolved acquisition sequence of excitation light, short-lifetime background fluorescence, and long-persistence fluorescence in this invention.

[0029] Figure 4 This is a schematic diagram illustrating the multi-time window acquisition and dynamic range expansion of the time domain of the present invention.

[0030] Figure 5 This is a schematic diagram of the probe set structure of the high-density four-probe step-by-step resequencing gene chip of the present invention.

[0031] Figure 6 This is a schematic diagram of the hardware structure of the reading system of the present invention.

[0032] Figure 7 This is a schematic diagram of the data processing and probe group interpretation process of the present invention.

[0033] Explanation of reference numerals in the attached figures

[0034] 10-Gene chip; 11-Probe array; 12-Probe group; 13-Probe site; 20-Reading system; 21-Pulsed excitation light source module; 22-Optical module; 23-Area array fluorescence detection module; 24-Timing control module; 25-Data processing module; 31-Biotinylated nucleotide; 32-Target nucleic acid product; 33-Streptavidin; 34-Long-afterglow fluorescent label. Wherein, tp represents the excitation pulse width, td represents the delay time from when the excitation light is turned off to when the acquisition window opens, tw represents the acquisition window width, and W1, W2, and W3 represent acquisition windows at different times.

Claims

1. A method for labeling and reading gene chips with long-persistence fluorescence time-resolved characteristics, Includes the following steps: S1. During the nucleic acid amplification or reverse transcription reaction of the sample to be tested, biotinylated nucleotides are added to the reaction system, and the biotinylated nucleotides are incorporated into the newly synthesized nucleic acid chain under the action of polymerase or reverse transcriptase to obtain the target nucleic acid product carrying biotin label. S2. Hybridize the target nucleic acid product with the probe array on the surface of the gene chip, so that the target nucleic acid product binds to the corresponding probe site; S3. Streptavidin coupled with a long-afterglow fluorescent marker binds to the biotin label on the target nucleic acid product, thereby generating a long-afterglow fluorescent signal at the corresponding probe site. S4. Irradiate the gene chip with a pulsed excitation light source, and after the pulsed excitation light source stops outputting excitation light, open the acquisition window again after a preset delay time to acquire the long afterglow fluorescence signal in a time-resolved manner. S5. Perform background correction, intensity extraction or fusion processing on the acquired time-resolved fluorescence signals, and generate at least one of the following based on the time-resolved fluorescence signal intensity of each probe site, probe site coordinates and probe array design information: target nucleic acid detection result and interpretation result.

2. The method according to claim 1, characterized in that, The biotinylated nucleotide is a biotin-labeled nucleotide that can be recognized by polymerase or reverse transcriptase and incorporated into a newly synthesized nucleic acid chain.

3. The method according to claim 2, characterized in that, The biotinylated nucleotides include biotin-labeled uracil deoxynucleotides, biotin-labeled cytosine deoxynucleotides, Biotin-11-dUTP, Biotin-11-dCTP, or combinations thereof.

4. The method according to claim 1, characterized in that, The long-afterglow fluorescent markers include lanthanide rare earth complex fluorescent markers that have a fluorescence lifetime in the microsecond to millisecond range after the excitation light is turned off.

5. The method according to claim 1, characterized in that, The streptavidin conjugated with a long-afterglow fluorescent marker is added before, during, or after hybridization of the target nucleic acid product with the probe array.

6. The method according to claim 1, characterized in that, The same target nucleic acid product carries multiple biotin-labeled sites, and the multiple biotin-labeled sites are respectively bound to multiple streptavidin molecules coupled with long-afterglow fluorescent labels.

7. The method according to claim 1, characterized in that, The excitation time of the pulsed excitation source is from 1 μs to 100 μs.

8. The method according to claim 1, characterized in that, The acquisition window opens after a delay of 1 μs to 1000 μs after the excitation light is turned off, and the acquisition window width is 10 μs to 10 ms. The delay time and acquisition window width are set according to the fluorescence lifetime of the long-afterglow fluorescent marker, the background decay time of the chip substrate, the response characteristics of the detection module, and the target signal intensity.

9. The method according to claim 1, characterized in that, Multiple acquisition windows are set after the same excitation to acquire long-afterglow fluorescence signals at different decay stages. Based on the signal saturation state, signal-to-noise ratio, or background level of different acquisition windows, the signals obtained from multiple acquisition windows are weighted, superimposed, replaced, corrected, or fused to expand the detection dynamic range.

10. The method according to claim 1, characterized in that, The gene chip is a high-density targeted resequencing gene chip. The probe array includes multiple probe sets, each probe set corresponding to a base position to be read in the target reference sequence. Each probe set includes at least four probe sites, and each of the at least four probe sites is respectively fixed with nucleic acid probes targeting different candidate bases.

11. The method according to claim 10, characterized in that, The probe array includes one or more of the following features: the target reference sequence positions corresponding to adjacent probe groups are moved sequentially according to a step size of one or more bases; and a sense probe group and an antisense probe group are set for the same base position to be read.

12. The method according to claim 10, characterized in that, Based on the time-resolved fluorescence signal intensity differences at different probe sites within the same probe group, the base type, mutation state, or genotyping result of the corresponding base position to be interpreted is determined. Specifically, the fluorescence intensity of each candidate probe site within the same probe group is subjected to background subtraction and normalization. The candidate probe with the highest normalized intensity and which meets the preset discrimination condition with the second highest normalized intensity is selected as the main interpretation result. If the preset discrimination condition is not met, a low confidence result or undetermined site is output.

13. The method according to any one of claims 1 to 11, characterized in that, The target nucleic acid detection results are used for detection of preset pathogen target libraries, drug resistance gene sites, pathogen mutation sites, or typing marker sites.

14. A long-persistence fluorescence time-resolved gene chip reading system, characterized in that, include: Gene chips are used to immobilize probe arrays; A pulsed excitation light source module is used to provide pulsed excitation light to the gene chip; An optical module is used to guide excitation light to the gene chip and collect the long-afterglow fluorescence signal emitted by the gene chip; An array fluorescence detection module is used to acquire the long-persistence fluorescence signal within the acquisition window; A timing control module is connected to the pulse excitation light source module and the area array fluorescence detection module respectively. The timing control module is configured to output an exposure trigger signal to the area array fluorescence detection module after a preset delay time after the pulse excitation light source module stops outputting excitation light, so that the area array fluorescence detection module delays opening the acquisition window. The data processing module is used to generate at least one of the following based on probe site coordinates, time-resolved fluorescence signals, and probe array design information: target nucleic acid detection results and interpretation results.

15. The reading system according to claim 14, characterized in that, The pulsed excitation light source module includes an ultraviolet LED, a violet LED, a semiconductor laser, or a combination thereof; the area array fluorescence detection module includes an industrial monochrome CMOS camera, a CCD image sensor, an sCMOS image sensor, or other area array image sensors; the optical module includes an excitation filter, a dichroic mirror, an emission filter, and an imaging lens, wherein the emission filter is used to transmit the emission band of the long-persistence fluorescent marker and suppress the excitation band.

Images