Near-infrared fluorescence sensor, near-infrared fluorescence sensor array and preparation method and application of near-infrared fluorescence sensor array

Abstract

The invention discloses a near-infrared fluorescence sensor, a near-infrared fluorescence sensor array and a preparation method and application of the near-infrared fluorescence sensor array, and belongs to the technical field of biological medicine. The composite material comprises a single-walled carbon nanotube, a single-stranded DNA (deoxyribonucleic acid) and hydrogel, the single-stranded DNA is connected and modified on the surface of the single-walled carbon nanotube, and the single-walled carbon nanotube modified with the single-stranded DNA is fixed in the hydrogel. Bacterial metabolites enter the hydrogel and influence the structure of the single-stranded DNA, so that the fluorescence intensity of the single-walled carbon nanotube is influenced. A plurality of near-infrared fluorescence sensors form an array to quickly capture component change caused by growth of bacteria in an object to be detected, and respond to fluorescence signal change, so as to visualize whether the components change or not. Whether bacteria grow or not is judged through response information of the multiple sensors, so that the bacteria are rapidly recognized, and the method has the advantages of being high in sensitivity, short in response time and small in needed sample size.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a near-infrared fluorescent sensor, a near-infrared fluorescent sensor array, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Sterility testing is a method used to check whether sterile drugs, biological products, medical devices, raw materials, excipients and other varieties required by the pharmacopoeia are sterile. It is a necessary method to ensure the quality of drugs and the safety of clinical operations. However, the sterility testing methods specified in the pharmacopoeias of various countries are mostly to observe the turbidity of the culture medium after 14 days of bacterial culture after pre-treatment of the test product. The bacterial enrichment process is time-consuming, and the accuracy and reliability of the results will also be affected by the subjective factors of the test personnel. For innovative drugs, the 14-day inspection and release method is a limiting factor for its inspection and release. Not only is it not conducive to the rapid discovery of microbial contamination risks during the production process, but for drugs with a short shelf life such as cell therapy drugs, it is even impossible to complete the inspection before the drug is used. Therefore, the development and utilization of rapid and sensitive sterility detection methods are of great significance for ensuring drug quality, establishing standards, and improving drug innovation standards.

[0004] Existing technologies that can be applied to sterility detection include flow cytometry (FCM), solid phase cell counting (SPC), real-time fluorescence quantitative PCR (Qμantitative Real-time PCR), etc., but the operation of these methods is complicated and cumbersome, and some of them have operating cost problems, making it difficult to achieve popular application. Patent CN110726840A discloses a DNA-two-dimensional material array sensor, including two-dimensional nanomaterials, DNA and fluorescent groups; the fluorescent groups are labeled at the 5' and / or 3' end of the DNA; the DNA is combined with the two-dimensional nanomaterial, so that the fluorescent groups labeled on the DNA are quenched. Microorganisms and two-dimensional nanomaterials undergo non-specific competitive reactions, and the microorganisms combine with the two-dimensional nanomaterials, so that the DNA is separated from the two-dimensional nanomaterials, and the fluorescent groups emit fluorescence. Because it is used to detect the types of bacteria, it is necessary to create fingerprints of multiple known microorganisms, and the more fingerprints of microorganisms are constructed, the more accurate the detection results are, and its operation is complicated and requires a large amount of samples. Therefore, exploring and innovating rapid bacterial detection technology, clarifying the endpoint of sterility testing, making up for the shortcomings of current sterility testing methods, shortening detection time, and improving detection sensitivity, accuracy, and automation are important measures to improve the production efficiency of sterile preparations and ensure their safety. Summary of the invention

[0005] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a near-infrared fluorescence sensor, a near-infrared fluorescence sensor array and a preparation method and application thereof. The near-infrared fluorescence sensor array provided by the present invention has the characteristics of high sensitivity, short response time, small sample amount required, and low preparation and operation cost.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A first aspect of the present invention provides a near-infrared fluorescence sensor, comprising: single-walled carbon nanotubes, single-stranded DNA (ssDNA) and hydrogel;

[0008] The single-stranded DNA is connected and modified on the surface of the single-walled carbon nanotube, and the single-walled carbon nanotube modified with the single-stranded DNA is fixed in the hydrogel.

[0009] The second aspect of the present invention provides a method for preparing the above-mentioned near-infrared fluorescence sensor, comprising the following steps:

[0010] The single-stranded DNA and the single-walled carbon nanotubes are mixed, dissolved in a solvent, and dispersed evenly at low temperature; after the dispersion is completed, the undispersed part and the unconnected single-stranded DNA are removed to obtain an optical sensor dispersion liquid;

[0011] The obtained optical sensor dispersion, polyethylene glycol diacrylate, polyethylene glycol, a photoinitiator and water are mixed and cross-linked to obtain a near-infrared fluorescent sensor.

[0012] The third aspect of the present invention provides a near-infrared fluorescent sensor array, comprising a plurality of the above-mentioned near-infrared fluorescent sensors or near-infrared fluorescent sensors prepared by the above-mentioned preparation method, wherein the single-stranded DNA types in at least two near-infrared fluorescent sensors are different.

[0013] A fourth aspect of the present invention provides an application of the above-mentioned near-infrared fluorescence sensor or the above-mentioned near-infrared fluorescence sensor array in sterility detection.

[0014] A fifth aspect of the present invention provides a method for detecting bacteria, comprising the following steps:

[0015] Determine the fluorescence intensity of the above-mentioned near-infrared fluorescence sensor array, which is recorded as the initial fluorescence intensity I0; respectively add the test solution to each near-infrared fluorescence sensor, let it stand until the test solution is absorbed, and then detect the fluorescence intensity again, which is recorded as I;

[0016] The fluorescence intensity of the near-infrared fluorescence sensor array is measured, which is recorded as the initial fluorescence intensity I0; a control solution is added to each near-infrared fluorescence sensor, and the control solution is allowed to stand until the control solution is absorbed, and the fluorescence intensity is measured again, which is recorded as I1;

[0017] The relative value of fluorescence intensity change was used for result analysis, i.e. (I-I0) / I0, (I1-I0) / I0; principal component cluster analysis was used to evaluate the distinction between the experimental group and the control group.

[0018] The beneficial effects of the present invention are:

[0019] The present invention is based on the change of growth medium components caused by the metabolism of bacteria during growth, and uses SWCNT and multiple ssDNA to construct sensors, and uses gel to fix and prepare sensor arrays. The array has the advantages of simple preparation, good stability and responsiveness, and is easy to operate. By dropping the sample solution to be tested and the sterile sample solution on the array to measure the difference in their fluorescence response, it can quickly identify whether the sample is contaminated with bacteria, and the sample consumption is small. The array can be used to quickly and accurately determine whether there is bacterial growth during the bacterial growth process of the sterile test medium, shortening the culture time. It can detect 10 bacteria after a short culture of 10 minutes. 2 The array can be used to detect whether sterile water for injection has bacterial contamination, without using culture medium to enrich the bacteria, and can detect as low as 10 3 The array can respond to and identify bacteria with CFU / mL. And because the time from sampling to completion of the test is no more than 2 minutes, bacteria in the air will not interfere with the test. Using this array, the sterility test process of sterile preparations can be accelerated in actual production, thereby speeding up their release. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 PBS dispersions of seven optical sensors prepared in Example 1 of the present invention, from left to right (GC) 15 -SWCNT, (C) 30 -SWCNT, (GT)5-SWCNT, (GT) 10 -SWCNT, (GT) 15 -SWCNT, (GT) 20 -SWCNT, (GT) 30 -SWCNT sensor dispersion;

[0022] Figure 2 Atomic force microscope (AFM) images of 7 optical sensors prepared in Example 1 of the present invention;

[0023] Figure 3 The UV-visible-near-infrared absorption spectra of 7 kinds of optical sensor dispersions prepared in Example 1 of the present invention;

[0024] Figure 4 The fluorescence spectra of the 7 optical sensor dispersions prepared in Example 1 of the present invention before and after pH changes;

[0025] Figure 5 The morphology characterization of the near-infrared fluorescence sensor (i.e., gel sensor) prepared in Example 2 of the present invention, wherein a is a near-infrared fluorescence sensor; b is a scanning electron microscope (SEM) imaging of the near-infrared fluorescence sensor; c is an observation of the fluorescence intensity change of 7 near-infrared fluorescence sensors using an inverted fluorescence microscope;

[0026] Figure 6 The near-infrared fluorescence sensor array (a) prepared in Example 3 of the present invention and its wide-field fluorescence imaging (b);

[0027] Figure 7 The near-infrared fluorescence sensor array prepared in Example 3 of the present invention is used for high-abundance ATCC Escherichia coli liquid (order of magnitude 10 8 CFU / mL) and sterile culture medium; a is the response of each sensor at different time points; b is the cluster analysis result of high-abundance bacterial liquid culture for 10 minutes and sterile culture medium;

[0028] Figure 8 The logarithm of the near-infrared fluorescence sensor array prepared in Example 3 of the present invention is 1×10 3 CFU / mL of bacterial solution cultured for 10 minutes and then differentiated from sterile culture medium; a is the 6 tubes of bacterial solution inoculated after the strain was activated once (10 3 CFU / mL) and the cluster analysis of sterile culture medium; b is the 6 bacterial solutions (10 3 CFU / mL), each portion was divided into 3 tubes for cluster analysis with sterile culture medium after culturing for 10 min;

[0029] Fig. 9 The logarithm of the near-infrared fluorescence sensor array prepared in Example 3 of the present invention is 10 2 CFU / mL of bacterial solution after 10 min of culture and differentiation from sterile culture medium;

[0030] Fig.10 The near-infrared fluorescence sensor array prepared in Example 3 of the present invention can be used to identify bacteria in sterile water for injection; wherein a is the ratio of the amount of sterile water for injection to the amount of bacteria introduced into the sterile water for injection.3 CFU / mL (left), 10 5 CFU / mL(Medium), 10 7 CFU / mL (right) Cluster analysis of ATCC Escherichia coli in sterile water for injection; b is the cluster analysis of sterile water for injection contaminated by airborne bacteria and sterile water for injection; c is the cluster analysis of sterile water for injection uncontaminated group, bacteria contaminated group and initial sterile water for injection; d is the result of bacterial culture of sterile water for injection uncontaminated group and bacteria contaminated group;

[0031] Fig.11 Analysis of the repeatability (a) and the repeatability (b) of the response of the near-infrared fluorescence sensor array prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] In view of the needs and existing problems of sterility testing of sterile products, and taking into account the simplicity of operation to minimize the impact of differences in experimental operations among operators, the present invention aims to prepare a fluorescent sensor array for rapid bacterial detection.

[0033] A typical embodiment of the present invention provides a near-infrared fluorescence sensor, comprising: single-walled carbon nanotubes, single-stranded DNA and hydrogel;

[0034] The single-stranded DNA is connected and modified on the surface of the single-walled carbon nanotube, and the single-walled carbon nanotube modified with the single-stranded DNA is fixed in the hydrogel.

[0035] Single-walled carbon nanotubes (SWCNTs) are a type of nanomaterial that is composed entirely of carbon atoms. Their geometric structure can be viewed as a hollow cylinder formed by the curling of graphene sheets. Under a specific excitation wavelength, single-walled carbon nanotubes can emit fluorescence in the near-infrared region II (900-1700nm), which has good penetration and low biological background fluorescence. After appropriate surface modification, not only can their dispersion in aqueous solution be improved, but their fluorescence can also be changed by the regulation of specific external factors, thus playing the role of a functional optical sensor.

[0036] Single-stranded DNA (ssDNA) is a hydrophilic biomolecule with a rich and diverse sequence library. SWCNT modified with ssDNA has good hydrophilicity and colloidal stability and can be stored in aqueous solution for a long time. The fluorescent sensor obtained after modification has a certain responsiveness to a variety of substances, and the SWCNT complexes obtained after ssDNA modification of different lengths and sequences have different fluorescence response characteristics. In bacterial detection, near-infrared fluorescent sensors can quickly capture the changes in various components (i.e., bacterial metabolites) in the test object caused by bacterial growth. The changes in components affect the surface interaction between ssDNA and SWCNT, and then affect the fluorescence intensity of SWCNT. In bacterial detection, ssDNA and SWCNT do not need to be separated. The interaction between the two changes due to environmental influences, causing the sensor to respond to changes in fluorescence signals, and the detection results are accurate.

[0037] Furthermore, the present invention fixes the single-walled carbon nanotubes modified with ssDNA in the hydrogel, which, on the one hand, improves the stability of the near-infrared fluorescence sensor, improves its repeatability, and is easy to store and use; on the other hand, the hydrogel plays a barrier role, preventing bacteria from contacting the single-walled carbon nanotubes modified with ssDNA, preventing them from affecting the structure of ssDNA, and improving the accuracy of the detection results; the third aspect is that the hydrogel does not reflect light during fluorescence intensity detection, making the fluorescence intensity detection result more accurate. If the hydrogel is not used and the fluorescence intensity of the solution is directly detected, the fluorescence detection result will be inaccurate due to the reflection of the aqueous solution, affecting the accuracy of the experimental results.

[0038] It should be noted that although there are only four types of deoxynucleotides (A, T, C, G) that make up ssDNA molecules and only two types of pairing methods (AT and CG), their number can be tens of thousands. More importantly, the arrangement order of base pairs can vary, thus determining the diversity of ssDNA molecules. Therefore, the diversity of ssDNA molecules provides larger and more diverse characteristic signal collection units for sterility detection methods.

[0039] In some examples of this embodiment, in order to better combine with the single-walled carbon nanotube, the base of the ssDNA is selected from one or more of G, C and T.

[0040] Preferably, the base arrangement sequence and base number of the ssDNA are not specifically limited, provided that the ssDNA does not specifically bind to bacteria.

[0041] Preferably, in order to better combine with single-walled carbon nanotubes and improve the accuracy of the detection result, the nucleic acid sequence of the ssDNA is any one of the sequences shown in SEQ ID NO: 1-7. The specific information of the sequences shown in SEQ ID NO: 1-7 is as follows.

[0042]

[0043] In some examples of this embodiment, the hydrogel is cross-linked by polyethylene glycol diacrylate (PEG-DA) and polyethylene glycol (PEG).

[0044] It should be noted that the pore size of the hydrogel is smaller than the volume of bacteria, and bacterial metabolites enter the hydrogel and affect the structure of ssDNA, thereby affecting the fluorescence intensity of single-walled carbon nanotubes. The pore size of the hydrogel is smaller than the volume of bacteria, but it is not the smaller the better. The smaller the pore size, the denser the gel network, which makes it difficult for bacterial metabolites in the test object to enter, affecting the test results. It has been proved by multiple experiments that the hydrogel cross-linked with PEG-DA and PEG-6000 is most suitable for fixing single-walled carbon nanotubes modified with ssDNA. The average molecular weight of the PEG-DA is 680-720.

[0045] More preferably, the hydrogel is obtained by mixing PEG-DA (average molecular weight ≈700), PEG-6000, single-walled carbon nanotubes modified with ssDNA, a photoinitiator and water, and then cross-linking and curing under ultraviolet light.

[0046] The photoinitiator includes, but is not limited to, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropenone, 2,4,6 (trimethylbenzoyl) diphenylphosphine oxide, and 2-phenylbenzyl-2-dimethylamine-1-(4-morpholinobenzylphenyl) butanone.

[0047] In another typical embodiment of the present invention, a method for preparing the above-mentioned near-infrared fluorescence sensor is provided, comprising the following steps:

[0048] After mixing ssDNA and single-walled carbon nanotubes, dissolving them in a solvent and dispersing them uniformly at low temperature; after dispersion is completed, removing the undispersed part and unconnected ssDNA to obtain an optical sensor dispersion;

[0049] The obtained optical sensor dispersion, polyethylene glycol diacrylate, polyethylene glycol, a photoinitiator and water are mixed and cross-linked to obtain a near-infrared fluorescent sensor.

[0050] In some examples of this embodiment, the mass ratio of the ssDNA to the single-walled carbon nanotubes is (0.9-1.1):(0.9-1.1), preferably 1:1.

[0051] In some examples of this embodiment, the solvent includes but is not limited to PBS buffer.

[0052] In some examples of this embodiment, the low temperature is -5°C-5°C, preferably an ice bath environment.

[0053] In some examples of this embodiment, the dispersion is uniform at low temperature, and in order to improve the dispersion effect, the dispersion can be performed with the aid of tip ultrasound. For example, a handheld 3mm probe tip ultrasound can be used to connect ssDNA to SWCNT under the condition of 20% power, and the probe is about 5mm below the liquid surface. The ultrasound process is performed in a manner of working for 2s and stopping for 1s, with a total working time of 80min, and the entire modification dispersion process is performed in an ice bath.

[0054] In some examples of this embodiment, the undispersed part is removed by centrifugation. For example, the obtained dispersion can be centrifuged three times at a centrifugal force of 16,100×g, each time for 60 minutes, to remove the undispersed matter.

[0055] In some examples of this embodiment, unattached ssDNA is removed by ultrafiltration and centrifugation. For example, 80% of the supernatant after the dispersion is removed can be taken to a 100 kDa ultrafiltration tube, centrifuged at 12,000 × g for 10 min, and then 200 μL PBS is added to resuspend the retentate, and centrifuged at 12,000 × g for 10 min. The process of adding PBS is repeated 3 times to remove unattached ssDNA, and the optical sensor is eluted and retained in the ultrafiltration tube.

[0056] It should be noted that the optical sensor precipitates that are aggregated and difficult to disperse due to ultrafiltration also need to be removed. The optical sensor after ultrafiltration can be resuspended in 400 μL PBS and centrifuged at 16,100×g for 30 minutes to remove the sensor precipitates that are aggregated and difficult to disperse due to ultrafiltration. The first 80% of the supernatant is retained to obtain the optical sensor dispersion.

[0057] In some examples of this embodiment, the obtained optical sensor dispersion, PEG-DA, PEG, a photoinitiator and water are mixed, and cross-linked and cured under ultraviolet light to obtain a near-infrared fluorescent sensor.

[0058] In order to improve the accuracy of the detection result, the average molecular weight of the PEG-DA is 680-720, the PEG is PEG-6000, and the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropenone.

[0059] Preferably, the mass ratio of PEG-DA to PEG-6000 is (0.9-1.1):(0.9-1.1), the amount of photoinitiator is 0.4%-0.5% of the mass of PEG-DA. The amount ratio of PEG-DA to water is 199-200 mg:1.5-2.0 mL, preferably 199-200 mg:1.8-1.9 mL.

[0060] In some examples of this embodiment, in the near-infrared fluorescence sensor, the final concentration of the single-walled carbon nanotubes satisfies that the absorbance of the near-infrared fluorescence sensor at 990nm is 0.18-0.22 (preferably 0.2); and the connection efficiency of the single-stranded DNA on the single-walled carbon nanotubes is 20-55%.

[0061] Preferably, the single-stranded DNA of the sequence shown in SEQ ID NO: 1 ((GC) 15 ) has a connection efficiency of 26-27% (preferably 26.35%) on single-walled carbon nanotubes; the single-stranded DNA of the sequence shown in SEQ ID NO: 2 ((C) 30 ) has a connection efficiency of 22-23% (preferably 22.12%) on single-walled carbon nanotubes; the connection efficiency of the single-stranded DNA of the sequence shown in SEQ ID NO: 3 ((GT)5) on single-walled carbon nanotubes is 36-37% (preferably 36.25%); the single-stranded DNA of the sequence shown in SEQ ID NO: 4 ((GT) 10 ) has a connection efficiency of 43-44% (preferably 43.45%) on single-walled carbon nanotubes; the single-stranded DNA of the sequence shown in SEQ ID NO: 5 ((GT) 15 ) has a connection efficiency of 44-45% (preferably 44.10%) on single-walled carbon nanotubes; the single-stranded DNA of the sequence shown in SEQ ID NO: 6 ((GT) 20 ) has a connection efficiency of 51-52% (preferably 51.12%) on single-walled carbon nanotubes; the single-stranded DNA of the sequence shown in SEQ ID NO: 7 ((GT) 30 ) on single-walled carbon nanotubes with a connection efficiency of 41-42% (preferably 41.65%).

[0062] It should be noted that the preparation methods of near-infrared fluorescent sensors prepared from different ssDNAs are the same, and only the type of ssDNA needs to be changed during the preparation.

[0063] A third typical embodiment of the present invention provides a near-infrared fluorescence sensor array, comprising a plurality of the above-mentioned near-infrared fluorescence sensors or near-infrared fluorescence sensors prepared by the above-mentioned preparation method, wherein the ssDNA types in at least two near-infrared fluorescence sensors are different.

[0064] The present invention is based on the fact that the composition of the growth medium will change due to the metabolism of the bacteria during growth. Single-walled carbon nanotubes (SWCNT) are used as the central material. After the surface is modified by different ssDNA, a fluorescent sensor with different response properties is prepared. After being fixed with the help of hydrogel, a near-infrared fluorescent sensor is prepared, and then a near-infrared fluorescent sensor array is prepared. In bacterial detection, an array composed of multiple near-infrared fluorescent sensors can quickly capture the changes in multiple components (i.e., bacterial metabolites) in the test object caused by bacterial growth, and respond to the changes in fluorescent signals. The array can be used to convert the changes in the small components of the medium into different degrees of changes in fluorescent signals, that is, to "visualize" whether the components have changed. The array is composed of multiple different universal response sensors, so even for weak material changes, it can be reflected because there are multiple different sensors to capture different changes in fluorescent signals. Whether there is bacterial growth is determined by the response information of multiple sensors, thereby realizing rapid identification of bacteria. The array has the characteristics of high sensitivity, short response time, small sample size, and low preparation and operation costs.

[0065] It should be noted that the near-infrared fluorescent sensor array of the present invention is used for qualitative detection of bacteria, and can be prepared into a bacteria detection kit to achieve rapid bacteria detection. In sterile detection, detection can be performed after culturing for 10 minutes, with short detection time and high sensitivity.

[0066] Preferably, the number of sensors in the near-infrared fluorescence sensor array may be a positive integer of 2-100, and at least one of the base arrangement order or base number of ssDNA in each near-infrared fluorescence sensor array is different.

[0067] Specifically, the number of the near-infrared fluorescence sensors is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100.

[0068] Since the ssDNA changes the base sequence, base number, and different secondary structures, the near-infrared fluorescent sensor has high scalability and can be combined into many different near-infrared fluorescent sensors. For example, different near-infrared fluorescent sensors can be obtained by selecting ssDNA with different base sequences; different near-infrared fluorescent sensors can be obtained by selecting ssDNA with different base numbers.

[0069] Preferably, the near-infrared fluorescent sensor array is composed of 7 near-infrared fluorescent sensors, and the ssDNA in the 7 near-infrared fluorescent sensors are of different types, and the sequences of the ssDNA in the 7 near-infrared fluorescent sensors are respectively shown in SEQ ID NOs: 1-7.

[0070] A fourth typical embodiment of the present invention provides an application of the above-mentioned near-infrared fluorescence sensor or the above-mentioned near-infrared fluorescence sensor array in sterility detection.

[0071] The array is composed of multiple different universal response sensors. Even for weak material changes, different fluorescence signal changes will be reflected because multiple different sensors are used to capture them. The response information of multiple sensors is used to determine whether bacteria are growing, thereby achieving rapid identification of bacteria. The array has the characteristics of high sensitivity, short response time, small sample volume, and low preparation and operation costs. Therefore, it has broad application prospects in sterility testing.

[0072] In some examples of this embodiment, the application includes rapid judgment of the end point of culture medium bacterial enrichment and rapid judgment of the bacterial contamination of sterile water for injection, and the rapid judgment is to perform the test after 8-12 minutes of cultivation to obtain the test result.

[0073] In some examples of this embodiment, the bacteria include but are not limited to Escherichia coli.

[0074] A fifth typical embodiment of the present invention provides a method for detecting bacteria, comprising the following steps:

[0075] Determine the fluorescence intensity of the above-mentioned near-infrared fluorescence sensor array, which is recorded as the initial fluorescence intensity I0; respectively add the test solution to each near-infrared fluorescence sensor, let it stand until the test solution is absorbed, and then detect the fluorescence intensity again, which is recorded as I;

[0076] The fluorescence intensity of the near-infrared fluorescence sensor array is measured, which is recorded as the initial fluorescence intensity I0; a control solution is added to each near-infrared fluorescence sensor, and the control solution is allowed to stand until the control solution is absorbed, and the fluorescence intensity is measured again, which is recorded as I1;

[0077] The relative value of fluorescence intensity change was used for result analysis, i.e. (I-I0) / I0, (I1-I0) / I0; principal component cluster analysis was used to evaluate the distinction between the experimental group and the control group.

[0078] The present invention successfully prepared a fluorescent sensor array for rapid bacterial detection, which can detect 10 2At the same time, the detection of sterile water for injection can detect as low as 10 CFU / mL of Escherichia coli without culture medium enrichment. 3 The array can respond to bacteria with CFU / mL. And because the time from sampling to completion of the test is no more than 2 minutes, bacteria in the air will not interfere with the test. Using this array, the sterility test process of sterile preparations can be accelerated in actual production, thereby speeding up their release.

[0079] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0080] The reagents used in the following examples are all commercially available products. The 7 kinds of ssDNA in the following examples are respectively marked as follows:

[0081] GCGCGCGCGCGCGCGCGCGCGCGCGCGCGC is marked with (GC) 15 ;

[0082] CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC marked with (C) 30 ;

[0083] GTGTGTGTGT is marked as (GT)5;

[0084] GTGTGTGTGTGTGTGTGTGTGT marked as (GT) 10 ;

[0085] GTGTGTGTGTGTGTGTGTGTGTGTGT is marked (GT) 15 ;

[0086] GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT is marked with (GT) 20 ;

[0087] GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT tagged (GT) 30 . (GC) 15 、(C) 30 、(GT)5、(GT) 10 , (GT) 15 , (GT) 20 , (GT) 30 They are ssDNA sequences and are all artificially synthesized.

[0088] Example 1

[0089] This embodiment is to prepare 7 kinds of optical sensor dispersions, and the specific steps are as follows:

[0090] Weigh 1 mg of single-walled carbon nanotube powder and ssDNA ((GC) 15 , 1 mg) were mixed, 2 mL of PBS buffer solution was added thereto, and ssDNA and SWCNT were connected by ultrasound with a handheld 3 mm probe tip at 20% power, with the probe penetrating about 5 mm below the liquid surface. The ultrasound process was carried out in a 2 s on, 1 s off mode, with a total working time of 80 min, and the entire modification and dispersion process was carried out in an ice bath.

[0091] The obtained dispersed solution was centrifuged three times at a centrifugal force of 16,100×g for 60 min each time to remove undispersed matter.

[0092] After three centrifugations, 80% of the supernatant obtained from the last centrifugation was taken to the inner tube of a 100 kDa ultrafiltration tube. After centrifugation at 12,000 × g for 10 min, 200 μL PBS was added to resuspend the retentate and centrifuged at 12,000 × g for 10 min. The process of adding PBS was repeated three times to remove unattached ssDNA. After elution, the sensor was retained in the ultrafiltration tube, and the (GC) obtained after each elution was retained. 15 Eluent.

[0093] Determined on NanoDrop micro-spectrophotometer (GC) 15 The concentration of the eluent was calculated according to (m 总 -c ssDNA ·v ssDNA ) / m 总 Calculated (GC) 15 The connection efficiency of ssDNA To determine the eluate (GC) 15 The concentration of v ssDNA is the total volume of the eluent; m 总 For use (GC) 15 total mass.

[0094] The sensor was resuspended in 400 μL PBS and centrifuged at 16,100 × g for 30 min to remove the sensor precipitate that was aggregated and difficult to disperse due to ultrafiltration. The first 80% of the supernatant was retained to obtain the optical sensor dispersion. The obtained optical sensor was referred to as (GC) 15 -SWCNT.

[0095] The preparation methods of the other six sensor dispersions were the same as above, and (C) 30-SWCNT, (GT)5-SWCNT, (GT) 10 -SWCNT, (GT) 15 -SWCNT, (GT) 20 -SWCNT, (GT) 30 -SWCNT 6 optical sensors. The connection efficiency of each single-stranded DNA is shown in Table 1.

[0096] The 7 optical sensor dispersions prepared in Example 1 are shown in Figure 1 , from left to right: (GC) 15 -SWCNT, (C) 30 -SWCNT, (GT)5-SWCNT, (GT) 10 -SWCNT, (GT) 15 -SWCNT, (GT) 20 -SWCNT, (GT) 30 - SWCNT optical sensor dispersion The optical sensor prepared in Example 1 has high dispersibility or stability, and no visible precipitation is generated during the solution preparation process.

[0097] Table 1 Ligation efficiency of single-stranded DNA

[0098]

[0099]

[0100] Performance characterization of seven optical sensors prepared in Example 1

[0101] 1. AFM imaging characterizes whether ssDNA is successfully modified on the SWCNT surface

[0102] (1) AFM sample preparation

[0103] Take 1 μL of each optical sensor dispersion, dilute it to 800 μL with ultrapure water, and use tip ultrasound to mix the system evenly to obtain each optical sensor AFM sample solution.

[0104] About 0.2 mg of SWCNT was weighed and dispersed using tip ultrasound at a power of 12% for 60 min, centrifuged at a centrifugal force of 16,100 × g for 60 min, and the precipitate was removed. The supernatant was the AFM sample solution of SWCNT.

[0105] Fix 8 clean mica sheets on the AFM iron sheet, and use transparent tape to tear off the surface mica sheet. Take 3 μL of each of the above 7 optical sensors and SWCNT AFM sample solutions, drop them on the clean mica sheets, and place them in a clean space to fully dry.

[0106] (2) AFM imaging

[0107] The samples on the mica sheet were measured in a gas phase environment using an atomic force microscope equipped with an RTESP-300 probe with a curvature radius of 10 nm to obtain the height of each sample. The scanning area was 1 μm and the scanning rate was 0.5 Hz. AFM images of SWCNT and each optical sensor are shown in Figure 2 .

[0108] According to the imaging results ( Figure 2 ) It can be seen that the diameter of SWCNT is about 1.1nm, and the diameters of the 7 optical sensors prepared are all significantly larger than 1.1nm. The height difference between the prepared complex and the untreated SWCNT shows that ssDNA is indeed adsorbed and entangled on SWCNT, that is, the 7 ssDNAs are successfully connected and modified to the SWCNT surface.

[0109] 2. UV-visible near-infrared absorption spectroscopy characterizes the uniform dispersion of the sensor in the system

[0110] 1 mL of each optical sensor dispersion prepared in Example 1 was transferred to a quartz cuvette, and the absorption spectrum of the optical sensor dispersion solution in the wavelength range of 400 nm to 1200 nm was measured using an ultraviolet-visible-near-infrared spectrophotometer.

[0111] The UV-visible-near-infrared absorption spectra of the seven optical sensor dispersions prepared in Example 1 are shown in Figure 3 .Depend on Figure 3 It was observed that each dispersion had characteristic absorption peaks of SWCNT near 560nm and 990nm. The existence of characteristic absorption peaks of SWCNT near 990nm and 560nm indicated that the seven optical sensors prepared in Example 1 could be uniformly dispersed in the system (PBS).

[0112] 3. Steady-state transient fluorescence spectroscopy characterizes the response performance of the sensor

[0113] Under the detection of UV-visible near-infrared spectrophotometer, PBS was used to adjust the absorbance of the 7 optical sensor dispersions prepared in Example 1 at 990nm to about 0.5, and then the original fluorescence spectrum of each sensor dispersion was obtained by scanning on the FLS1000 steady-state transient fluorescence spectrometer, with the scanning starting wavelength of 900nm, the cutoff wavelength of 1100nm, the step length of 1nm, and the excitation wavelength of 570nm. 20μL of 0.5M / L hydrochloric acid solution was added dropwise thereto, mixed evenly, and then the fluorescence spectrum was scanned again.

[0114] The fluorescence spectra of the seven optical sensors prepared in Example 1 are shown in Figure 3 .Depend on Figure 3It was observed that the fluorescence intensity of the seven optical sensors decreased after adding hydrochloric acid, that is, they all responded to the change in fluorescence intensity to the pH change, indicating that the seven optical sensors all have sensor response performance.

[0115] Example 2

[0116] This example uses the 7 optical sensor dispersions prepared in Example 1 to prepare 7 near-infrared fluorescence sensors. The specific steps are as follows:

[0117] (1) Preparation of photoinitiator solution

[0118] 6 mg of photoinitiator (2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropenone) was weighed, 500 μL of water was added thereto, and the mixture was stirred in a water bath at 60° C. for 20 min until it was completely dissolved.

[0119] (2) Prepare PEG-6000 solution

[0120] Weigh 1.92 g of PEG-6000 and fully dissolve it in 8 mL of PBS.

[0121] (3) Configuration and curing of near-infrared fluorescence sensor system

[0122] Weigh 200 mg of PEG-DA (average molecular weight ~700), add 83.2 μL of photoinitiator solution and 832.8 μL of PEG-6000, and finally add A 990 =0.5 (GT) 10 - 906 μL of SWCNT sensor dispersion, mixed thoroughly.

[0123] The prepared solution system was added dropwise to the glass bottom groove of the confocal dish, and cross-linked and cured in an ultraviolet cross-linking box. After curing, the solution was soaked and washed overnight with PBS to obtain a near-infrared fluorescence sensor (a gel sensor). Figure 5 (a), referred to as (GT) 10 -SWCNT near-infrared fluorescence sensor.

[0124] The preparation methods of the other six near-infrared fluorescence sensors are the same as (GT) 10 -SWCNT near-infrared fluorescence sensor.

[0125] Morphological characterization of the near-infrared fluorescence sensor prepared in Example 2

[0126] 1. SEM imaging characterization of the microscopic morphology of near-infrared fluorescence sensors

[0127] The microscopic morphology of the near-infrared fluorescence sensor prepared in Example 2 was observed using SEM imaging ( Figure 5(b)) It can be seen that it has a large pore size, which can allow the solution to penetrate from one side to the other side, and has the conditions for preparing to form an array in a container such as a well plate.

[0128] 2. Observation of the response performance of near-infrared fluorescence sensor using an inverted fluorescence microscope

[0129] The near-infrared fluorescence sensor prepared in the confocal dish was observed on an inverted fluorescence microscope. The sample (i.e., the near-infrared fluorescence sensor prepared in Example 2) was excited with 561 nm excitation light, and the excited fluorescence was detected with a XEVA near-infrared optimized InGaAs camera and imaged on the corresponding software. 40 μL of hydrochloric acid was dripped onto its surface, as Figure 5 As shown in (c), the fluorescence intensity of the gel was observed to change observably before and after the addition, indicating that after being prepared into a near-infrared fluorescence sensor, the sensor still retained its sensing performance.

[0130] Example 3

[0131] This embodiment is to prepare a near-infrared fluorescence sensor array, and the specific steps are as follows:

[0132] According to the configuration method of the gel solution system in step (3) of Example 2, 7 sensor gel solutions were configured and arranged in order in the Freedom EVO automated workstation. The program was set to drip each infrared fluorescence sensor solution into the 384-well plate in a specific order. In this example, 7 rows × 3 columns of small holes constitute a sensor array, where each row is a sensor, and each sensor is prepared 3 times. After the solution is added, the sensor array is placed in a UV crosslinking box. After crosslinking for 3 minutes, a near-infrared fluorescence sensor array is obtained. Figure 6 (a) shown.

[0133] Performance Characterization of the Near-Infrared Fluorescence Sensor Array Prepared in Example 3

[0134] 1. Wide-field fluorescence imaging of near-infrared fluorescent sensor arrays

[0135] The near-infrared fluorescence sensor array prepared in Example 3 was placed in a wide-field imaging system, a 561 nm laser was used to excite the sensor array, and the excited fluorescence was detected using a XEVA near-infrared optimized InGaAs camera and fluorescence imaging was performed on the corresponding software. The results are shown in FIG. Figure 6 (b) as shown.

[0136] 2. Application of near-infrared fluorescence sensor array in sterility detection - rapid determination of the end point of culture medium enrichment

[0137] (1) Research on the differentiation of bacterial liquid and sterile culture medium using near-infrared fluorescence sensor array

[0138] Dissolve 5g of LB liquid culture medium powder completely in 200mL of pure water and sterilize at 121℃ for 15min. Take 2mL of sterile LB liquid culture medium to a shaking tube and inoculate 20μL of ATCC25922 Escherichia coli -20℃ strain frozen liquid. Place the inoculated culture medium at 37℃ and culture it in a shaking incubator at 225rpm overnight.

[0139] Add 1 mL of overnight activated bacterial solution to a 250 mL Erlenmeyer flask containing 100 mL of sterile LB liquid medium and culture in a shaker at 37°C and 225 rpm. Take out 2 mL of bacterial solution every 5 minutes for the first 30 minutes, and then take out 2 mL of bacterial solution every 10 minutes for detection and bacterial count on the near-infrared fluorescence sensor array. Place the bacterial solution taken at each time point in an ice box and detect it on the near-infrared fluorescence sensor array in chronological order.

[0140] The original fluorescence intensity of the near-infrared fluorescence sensor array was measured by a fluorescence imaging system, recorded as I0; 20 μL of bacterial solution was added to each near-infrared fluorescence sensor hole, and the fluorescence intensity was detected again after standing for 2 minutes, recorded as I. The bacterial solution and sterile culture medium at each time point were measured according to the above method to obtain two groups of fluorescence values, I0 and I. Each time point and sterile culture medium were measured three times. In order to eliminate the influence of stray light on the fluorescence measurement results, the relative value of the fluorescence intensity change (i.e., (I-I0) / I0) was used to analyze the results. The PCA analysis method provided by MetaboAnalyst was used for the analysis. Cluster analysis was used to observe whether the fluorescence detection results of the bacterial solution and sterile culture medium at each time point (i.e., the value of (I-I0) / I0) could be separated to distinguish whether there was bacterial growth.

[0141] Figure 7 (a) is the response of each sensor at different time points. Figure 7 (b) is the PCA analysis result of high-abundance bacterial solution cultured for 10 minutes and sterile culture medium. The result analysis shows that after a short culture of 10 minutes, the bacterial solution can be distinguished from the sterile culture medium, indicating that the sensor array has the ability to distinguish the sterile culture medium from the bacterial solution.

[0142] (2) Research on the sensor array for distinguishing low-abundance bacterial solution and sterile culture medium

[0143] Dissolve 2.5g of LB liquid culture medium powder completely in 100mL of pure water and sterilize at 121℃ for 15min. Take 2mL of sterile LB liquid culture medium to a shaking tube and inoculate 20μL of ATCC25922 Escherichia coli -20℃ culture stock. Place the inoculated culture medium at 37℃, culture it in a shaking incubator at 225rpm overnight and dilute it with PBS to a concentration of 1×10 5CFU / mL.

[0144] Add 2 mL of culture medium to each of the 6 shaking tubes, inoculate 20 μL of diluted bacterial solution into each tube, and count the number of bacteria after colony counting to 1×10 3 CFU / mL. Place each shaking tube in a shaker at 37°C and 225 rpm for incubation.

[0145] The original fluorescence intensity of the infrared fluorescence sensor array was measured using a fluorescence imaging system, recorded as I0; 20 μL of bacterial solution was added to each near-infrared fluorescence sensor hole, and the fluorescence intensity was detected again after standing for 2 minutes, recorded as I. The bacterial solution and sterile culture medium at each time point were measured according to the above method to obtain two groups of fluorescence values, I0 and I, and each time point and sterile culture medium were measured three times. In order to eliminate the influence of stray light on the fluorescence measurement results, the relative value of the fluorescence intensity change (i.e., (I-I0) / I0) was used for result analysis.

[0146] The experimental results are as follows Figure 8 As shown in (a), analysis of the experimental results shows that after a short-term culture of 10 minutes, the low-abundance bacterial solution can be distinguished from the sterile culture medium.

[0147] The pH value of the sterile culture medium and the bacterial solution after 10 minutes of culture was measured using a pH meter. It was found that the pH values ​​of both fluctuated between 6.57 and 6.60, with no significant difference.

[0148] The absorbance of the sterile culture medium and the bacterial solution after 10 minutes of culture at 600 nm was measured using a UV-visible near-infrared spectrophotometer. The results are shown in Table 2. 600 There is also no difference.

[0149] Table 2

[0150]

[0151] To further verify the ability of the near-infrared fluorescence sensor array prepared in Example 3 to distinguish between sterile culture medium and low-abundance bacterial solution, the experiment was conducted again using bacterial solutions from different activated batches. Six tubes of E. coli-20 frozen solutions were activated respectively, and the six groups of bacterial solutions from different activated batches were diluted and inoculated into three tubes of 2 mL culture medium respectively, obtaining six groups of 18 bacterial solutions in total. The number of bacteria in each group was counted to confirm that the number of bacteria was 1×10 3 CFU / mL, each shaking tube was placed in a shaker at 37°C and 225 rpm for 10 min, and the determination was performed according to the determination method in “(1) Investigation of the differentiation of bacterial liquid and sterile culture medium by near-infrared fluorescence sensor array”.

[0152] The experimental results are shown in Figure 8(b) In the six groups of experiments, the fluorescence detection data of each bacterial solution can be distinguished from the detection data of the sterile culture medium, indicating that the array can detect the growth of bacteria in the above six experimental systems, proving that the detection performance of the array is accurate and stable.

[0153] (3) Determination of the detection limit of the sensor array at a fixed incubation time of 10 min

[0154] According to the method in “(1) Study on the differentiation of bacterial solution and sterile culture medium by near-infrared fluorescence sensor array”, the overnight activated bacterial solution was obtained and inoculated into 2 mL culture medium to obtain a fluorescence intensity of 1×10 2 6 tubes of bacterial solution with 1×10 CFU / mL and 1×10 CFU / mL were cultured at 37°C, 225 rpm in a shaker for 10 min and differentiated on the array.

[0155] The experimental results are as follows Fig. 9 As shown in the experimental results, the minimum distinguishable concentration under 10 min incubation time is 1×10 2 Bacterial liquid at the CFU / mL level.

[0156] 3. Application of near-infrared fluorescence sensor array in sterility detection - rapid determination of bacterial contamination in sterile water for injection

[0157] (1) Sensor array distinguishes ATCC Escherichia coli-contaminated water for injection from untreated sterile water for injection

[0158] Centrifuge 1 mL of overnight activated bacterial solution at 8000 rpm for 10 min, discard the supernatant, wash the bacterial precipitate three times with sterile injection water to completely remove the influence of the culture medium, and finally resuspend the bacterial precipitate with 1 mL of sterile injection water to obtain a dispersion.

[0159] The resuspended bacterial solution was diluted with sterile water for injection to obtain a solution containing 1×10 3 CFU / mL, 1×10 5 CFU / mL, 1×10 7 CFU / mL of sterile water for injection containing Escherichia coli. After each group of sterile water for injection containing bacteria was left to stand at room temperature for 30 minutes, it was distinguished from the untreated sterile water for injection on the array. The results were analyzed according to the method in "2. Application of near-infrared fluorescence sensor array in sterility detection - rapid judgment of the end point of culture medium enrichment".

[0160] The experimental results are as follows Fig.10 As shown in (a), each group with bacterial contamination can be distinguished from untreated sterile water for injection.

[0161] (2) Sensor array distinguishes between water for injection contaminated with foreign bacteria and untreated sterile water for injection

[0162] Take 10mL of sterile water for injection in a sterile 50mL centrifuge tube, expose it to air pollution for 30 minutes, divide it into shaking tubes, and let it stand overnight at room temperature. Take the water for injection after overnight culture and the untreated sterile water for injection on the sensor array for detection and differentiation, and analyze the results according to the method in "2. Application of near-infrared fluorescence sensor array in sterility detection-rapid judgment of culture medium enrichment end point".

[0163] The experimental results are as follows Fig.10 As shown in (b), the group exposed to bacterial contamination can be well distinguished from the uncontaminated injection water on the sensor array.

[0164] To further confirm the feasibility of the sensor array to distinguish between injection water contaminated by miscellaneous bacteria and sterile injection water. The sterile injection water was divided into two groups, E1 and E2, with 5 mL in each group. The E2 group was exposed to contamination for 30 minutes, and the E1 group was not treated. 1 mL of each group of injection water was taken in LB culture medium and cultured in a 37°C incubator. The rest was divided into shaking tubes and cultured overnight at room temperature.

[0165] Two groups of injection water after overnight culture and sterile injection water without any treatment were taken on the sensor array for detection and differentiation.

[0166] The experimental results are as follows Fig.10 As shown in (c), the experimental results show that the E2 group that can be distinguished from the initial sterile water for injection on the array has bacterial growth after 24 hours of culture, while the E1 group that cannot be distinguished still has no bacterial growth after a long period of culture. This shows that the array can directly identify whether the sterile water for injection is contaminated with bacteria without culture medium enrichment.

[0167] (3) Investigation of the preparation stability and response stability of near-infrared fluorescence sensor array

[0168] Six sensor arrays were prepared according to the method in Example 2, and the fluorescence intensity was observed in the fluorescence imaging system. Fig.11 (a).

[0169] 20 μL of bacterial solution after 10 minutes of culture was added to each well of the 6 sensor arrays, and the fluorescence intensity of the arrays before and after the addition was measured. The stability of the response of the near-infrared fluorescence sensor array was analyzed according to the method in "2. Application of near-infrared fluorescence sensor array in sterility detection - rapid judgment of the end point of culture medium enrichment". The experimental results are shown in Fig.11 (b).

[0170] The experimental results show that the near-infrared fluorescence sensor array is prepared by the method of the present invention, and the repeatability between groups is high, and the response also has good stability and repeatability.

[0171] Example 4

[0172] This embodiment is to prepare a near-infrared fluorescent sensor array. The specific steps are different from those of Embodiment 3 in that six optical sensor dispersions are prepared: (C) 30 -SWCNT, (GT)5-SWCNT, (GT) 10 -SWCNT, (GT) 15 -SWCNT, (GT) 20 -SWCNT, (GT) 30 -SWCNT. Six sensor gel solutions were prepared based on the six optical sensor dispersions. A sensor array was formed with 6 rows × 3 columns of small holes, where each row was a sensor, and each sensor was prepared three times. The remaining steps were consistent with those in Example 3.

[0173] Performance testing:

[0174] When the sensor array composed of the above six sensors is used to detect bacteria according to the steps in Example 3, for high-abundance bacterial liquid and the order of magnitude of 1×10 3 When distinguishing low-abundance bacterial liquid with CFU / mL from sterile culture medium, the separation effect is similar to that in Example 3, but for the order of magnitude of 1×10 2 For low-abundance bacterial liquid of CFU / mL, the gel sensor array cannot distinguish the bacterial liquid from the sterile culture medium.

[0175] For the detection of bacteria in sterile water for injection, only the introduced bacteria with an order of magnitude of 1×10 7 CFU / mL of Escherichia coli in sterile water for injection.

[0176] Example 5

[0177] This embodiment is to prepare a near-infrared fluorescent sensor array. The specific steps are different from those of Example 3 in that five optical sensor dispersions are prepared: (GT)5-SWCNT, (GT) 10 -SWCNT, (GT) 15 -SWCNT, (GT) 20 -SWCNT, (GT) 30 -SWCNT. Five sensor gel solutions were prepared based on the five optical sensor dispersions. A sensor array was formed with 5 rows × 3 columns of small holes, where each row was a sensor, and each sensor was prepared three times. The remaining steps were consistent with those in Example 3.

[0178] Performance testing:

[0179] When the sensor array composed of the above five sensors performs bacterial detection according to the steps in Example 3, it can show a separation effect similar to that in Example 3 when distinguishing high-abundance bacterial liquid from sterile culture medium. However, when detecting and distinguishing low-abundance bacterial liquid, the gel sensor array cannot distinguish the bacterial liquid from the sterile culture medium.

[0180] For bacterial contamination testing of sterile water for injection, it is impossible to distinguish between water for injection contaminated with Escherichia coli and sterile water for injection.

[0181] Example 6

[0182] This embodiment is to prepare a near-infrared fluorescence sensor array. The specific steps are different from those of Embodiment 3 in that four optical sensor dispersions are prepared: (GT) 10 -SWCNT, (GT) 15 -SWCNT, (GT) 20 -SWCNT, (GT) 30 -SWCNT. Four sensor gel solutions were prepared based on the four optical sensor dispersions. A sensor array was formed with 4 rows × 3 columns of small holes, where each row was a sensor, and each sensor was prepared three times. The remaining steps were consistent with those in Example 3.

[0183] Performance testing:

[0184] When the sensor array composed of the above four sensors performs bacterial detection according to the steps in Example 3, it can show a separation effect similar to that in Example 3 when distinguishing high-abundance bacterial liquid from sterile culture medium. However, when detecting and distinguishing low-abundance bacterial liquid, the gel sensor array cannot distinguish the bacterial liquid from the sterile culture medium.

[0185] For bacterial contamination testing of sterile water for injection, it is impossible to distinguish between water for injection contaminated with Escherichia coli and sterile water for injection.

[0186] Example 7

[0187] This embodiment is to prepare a near-infrared fluorescence sensor array. The specific steps are different from those of Embodiment 3 in that three optical sensor dispersions are prepared: (GT) 15 -SWCNT, (GT) 20 -SWCNT, (GT) 30 -SWCNT. Three kinds of sensor gel solutions were prepared based on the three optical sensor dispersions. A sensor array was formed with 3 rows × 3 columns of small holes, where each row was a sensor, and each sensor was prepared 3 times. The remaining steps were consistent with those in Example 3.

[0188] Performance testing:

[0189] When the sensor array composed of the above three sensors is used to detect bacteria according to the steps in Example 3, the detection results are not repeatable for high-abundance bacterial liquid. The three tubes of high-abundance bacterial liquid and sterile culture medium were tested three times, and there were both distinguishable and indistinguishable results.

[0190] For bacterial contamination testing of sterile water for injection, it is impossible to distinguish between water for injection contaminated with Escherichia coli and sterile water for injection.

[0191] Example 8

[0192] This embodiment is to prepare a near-infrared fluorescence sensor array. The specific steps are different from those of Example 3 in that two optical sensor dispersions are prepared: (GT) 20 -SWCNT, (GT) 30 -SWCNT. Two sensor gel solutions were prepared based on the two optical sensor dispersions. A sensor array was formed with 2 rows × 3 columns of small holes, where each row was a sensor, and each sensor was prepared 3 times. The remaining steps were consistent with those in Example 3.

[0193] Performance testing:

[0194] When the sensor array composed of the above two types of sensors is used to detect bacteria according to the steps in Example 3, the bacterial liquid and the sterile culture medium cannot be distinguished.

[0195] Comparative Example 1

[0196] This embodiment is for preparing a near-infrared fluorescent sensor array. The specific steps are different from those of Example 3 in that PEG-6000 is not added during the preparation of the gel sensor, and the remaining steps are consistent with those of Example 3.

[0197] Upon testing, it was found that no obvious pore size could be observed in the obtained near-infrared fluorescent sensor gel under SEM imaging at the same magnification as the near-infrared fluorescent sensor gel used in Example 3 or at a higher magnification, and therefore the near-infrared fluorescent sensor gel could not be used for sterility testing.

[0198] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A near-infrared fluorescence sensor, characterized in that: include: single-walled carbon nanotubes, single-stranded DNA, and hydrogels; The single-stranded DNA is connected and modified on the surface of the single-walled carbon nanotube, and the single-walled carbon nanotube modified with the single-stranded DNA is fixed in the hydrogel.

2. The near-infrared fluorescence sensor according to claim 1, characterized in that: The bases of the single-stranded DNA are selected from one or more of G, C and T; Preferably, the nucleic acid sequence of the single-stranded DNA is any one of the sequences shown in SEQ ID NOs: 1-7.

3. The near-infrared fluorescence sensor according to claim 1, characterized in that: The hydrogel is formed by cross-linking polyethylene glycol diacrylate and polyethylene glycol; Preferably, the hydrogel is formed by cross-linking polyethylene glycol diacrylate and polyethylene glycol 6000, and the average molecular weight of the polyethylene glycol diacrylate is 680-720.

4. A method for preparing a near-infrared fluorescence sensor according to any one of claims 1 to 3, characterized in that: The steps include: The single-stranded DNA and the single-walled carbon nanotubes are mixed, dissolved in a solvent, and dispersed evenly at low temperature; after the dispersion is completed, the undispersed part and the unconnected single-stranded DNA are removed to obtain an optical sensor dispersion liquid; The obtained optical sensor dispersion, polyethylene glycol diacrylate, polyethylene glycol, a photoinitiator and water are mixed and cross-linked to obtain a near-infrared fluorescent sensor.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the single-stranded DNA to the single-walled carbon nanotube is (0.9-1.1):(0.9-1.1); Preferably, the solvent comprises PBS buffer; Preferably, the low temperature is -5°C to 5°C.

6. The preparation method according to claim 4, characterized in that: In the near-infrared fluorescence sensor, the final concentration of the single-walled carbon nanotubes satisfies that the absorbance of the near-infrared fluorescence sensor at 990 nm is 0.18-0.22; the connection efficiency of the single-stranded DNA on the single-walled carbon nanotubes is 20-55%; Preferably, the connection efficiency of the single-stranded DNA of the sequence shown in SEQ ID NO: 1 on the single-walled carbon nanotube is 26-27%; the connection efficiency of the single-stranded DNA of the sequence shown in SEQ ID NO: 2 on the single-walled carbon nanotube is 22-23%; the connection efficiency of the single-stranded DNA of the sequence shown in SEQ ID NO: 3 on the single-walled carbon nanotube is 36-37%; the connection efficiency of the single-stranded DNA of the sequence shown in SEQ ID NO: 4 on the single-walled carbon nanotube is 43-44%; the connection efficiency of the single-stranded DNA of the sequence shown in SEQ ID NO: 5 on the single-walled carbon nanotube is 44-45%; the connection efficiency of the single-stranded DNA of the sequence shown in SEQ ID NO: 6 on the single-walled carbon nanotube is 51-52%; the connection efficiency of the single-stranded DNA of the sequence shown in SEQ ID NO: 7 on the single-walled carbon nanotube is 41-42%.

7. A near-infrared fluorescence sensor array, characterized in that: The invention comprises a plurality of near-infrared fluorescent sensors according to any one of claims 1 to 3 or near-infrared fluorescent sensors prepared by the preparation method according to any one of claims 4 to 6, wherein the single-stranded DNA types in at least two near-infrared fluorescent sensors are different.

8. The near-infrared fluorescence sensor array according to claim 7, characterized in that: The invention comprises 7 near-infrared fluorescent sensors, and the types of single-stranded DNA in the 7 near-infrared fluorescent sensors are different. The sequences of the single-stranded DNA in the 7 near-infrared fluorescent sensors are shown in SEQ ID NOs: 1-7 respectively.

9. Use of the near-infrared fluorescent sensor according to any one of claims 1 to 3 or the near-infrared fluorescent sensor array according to claim 7 or 8 in sterility detection; Preferably, the application includes rapid determination of the end point of bacterial enrichment in a culture medium and rapid determination of bacterial contamination in sterile water for injection, wherein the rapid determination is to perform a test after 8-12 minutes of culture to obtain a test result; Preferably, the bacteria include Escherichia coli.

10. A method for detecting bacteria, characterized in that: The steps include: Determine the fluorescence intensity of the near-infrared fluorescence sensor array according to claim 7 or 8, and record it as the initial fluorescence intensity I0; respectively add the test solution to each near-infrared fluorescence sensor, let it stand until the test solution is absorbed, and detect the fluorescence intensity again, which is recorded as I; Determine the fluorescence intensity of the near-infrared fluorescence sensor array according to claim 7 or 8, and record it as the initial fluorescence intensity I0; respectively add a control solution to each near-infrared fluorescence sensor, let it stand until the control solution is absorbed, and detect the fluorescence intensity again, and record it as I1; The relative value of fluorescence intensity change was used for result analysis, i.e. (I-I0) / I0, (I1-I0) / I0; principal component cluster analysis was used to evaluate the distinction between the experimental group and the control group.

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