A method for detecting a bio-orthogonal reaction immobilizing bacteria for ultra-sensitive fluorescent counting immunoassay

Abstract

This invention discloses a detection method for ultrasensitive fluorescence counting immunoassay using bioorthogonal reaction-immobilized bacteria, comprising the following steps: (1) transforming recombinant plasmids into *E. coli*, and inducing transcription into the T promoter using IPTG to induce *E. coli* to express green fluorescent protein; (2) dissociating Cu-MOF nanoparticles with ascorbic acid, allowing the azide groups of *E. coli* modified with azidoform to bind to the alkyne groups on alkyne-modified bovine serum albumin, thus immobilizing *E. coli* on a well plate; (3) amplifying the signal through the self-replication of luminescent *E. coli*, converting the concentration of the biomarker into fluorescent bright spots of fluorescent *E. coli*, thereby achieving quantitative detection. This invention has strong detection specificity, good stability and accuracy, and a detection limit lower than the clinical gold standard ELISA, allowing for visual detection of biomarker concentrations.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterials and laboratory medicine, specifically to a detection method for ultrasensitive fluorescence counting immunoassay using bioorthogonal reaction-immobilized bacteria. Background Technology

[0002] Despite the proliferation of various detection technologies, significant challenges remain in developing new techniques for detecting diseases in their early stages. Highly sensitive and accurate analysis of disease-related protein biomarkers in the early stages of diagnostic and treatment information feedback is crucial for disease prevention and control. Clinically, enzyme-linked immunosorbent assay (ELISA) is the gold standard method for specific protein assays. However, protein biomarkers are often present in complex biological environments and are highly sensitive to their surroundings; therefore, the common limit of detection (LOD) for ELISA is at the picomolar level, which is insufficient for detecting low concentrations of biomarkers.

[0003] In recent years, fluorescence counting has gradually become a powerful tool for developing ultrasensitive and reproducible biosensors. Calculating fluorescent bright spots within a specific area can be used to determine the quantity or concentration of an analyte. The yes / no measurement model overcomes the influence of intensity fluctuations and has shown outstanding potential in measuring low-concentration proteins. Organic fluorophore dyes or quantum dots (QDs) are commonly used as fluorescent probes for counting protein biomarkers. However, these fluorescent probes are prone to fluorescence quenching and require high-resolution imaging systems; the fluorescence signal is also difficult to amplify further for more sensitive detection. Therefore, there is a need to develop a new signal reporting method to achieve detection with high sensitivity, good stability, and ease of operation.

[0004] Fluorescent bacteria (EGFP-labeled E. coli) have attracted considerable attention as a live signal reporter due to their unique optical properties, high photostability, and low background, making them an ideal alternative to fluorescent dyes and quantum dots. Most significantly, their characteristic self-replication ability (fluorescent bacteria can reproduce one generation every 20–30 min at 37°C) can be used to amplify signals, thus enabling the detection of low-concentration biomarkers. However, fluorescent bacteria are too large to be directly used as probes in highly sensitive ELISA assays. Therefore, an efficient signal conversion method is urgently needed to convert the concentration of biomarkers into the number of fluorescent bacteria.

[0005] Currently, bioorthogonal reactions have attracted widespread attention due to their high selectivity, rapidity, and good biocompatibility, thus becoming a reliable signal transduction method. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a detection method for highly specific biological orthogonal reaction-fixed bacteria for ultrasensitive fluorescence counting immunoassay.

[0007] To address the problems of existing technologies, this invention provides the following technical solution: A method for detecting bacteria fixed by bioorthogonal reaction using ultrasensitive fluorescence counting immunoassay, comprising the following steps:

[0008] (1) pET-28a-EGFP recombinant plasmid was transformed into E. coli and IPTG was used to induce E. coli to express green fluorescent protein;

[0009] (2) Cu-MOF nanoparticles were dissociated with ascorbic acid to reduce Cu(II) to Cu(I). The azide group combined with the alkynyl group on the alkynylated bovine serum albumin. The azide group and the alkynyl group under the catalysis of Cu(I) underwent a click reaction to achieve the effect of immobilizing azide-modified Escherichia coli on a 96-well plate.

[0010] (3) The concentration of biomarkers is converted into fluorescent bright spots of fluorescent Escherichia coli by the self-replication of luminescent Escherichia coli, thereby realizing quantitative detection. The signal amplification effect is achieved by the proliferation of azidated Escherichia coli, and the signal amplification time is 20-30 min.

[0011] Further, in step (2), bovine serum albumin and N-propyne maleimide are mixed in PBS and concentrated by filtration using an ultrafiltration tube to obtain alkynylated bovine serum albumin BSA. The mass ratio of alkynylated bovine serum albumin BSA to N-propyne maleimide is 25:1.

[0012] Further, in step (2), KDO-N3 (3-deoxy-D-manno-2-octulose azide) specifically incorporates the azide group into the lipopolysaccharide on the E. coli membrane, thereby functionalizing it with azide; the azide group undergoes a click reaction with the alkyne group attached to the BSA already coated on the 96-well plate, and the E. coli is immobilized on the 96-well plate.

[0013] Furthermore, in step (2), Cu-MOF is obtained by reacting copper nitrate trihydrate with high temperature and pressure: PVP is dissolved in a mixed solvent of DMF and ethanol to prepare solution A; then copper nitrate trihydrate and 2-aminoterephthalic acid are dissolved in DMF to prepare solution B. After mixing solutions A and B, they are reacted in a high-pressure reactor at 100°C for 12 hours to obtain Cu-MOF.

[0014] Further, in step (2), the antibody that captures the cancer-specific antigen CEA is modified onto Cu-MOF nanoparticles. First, the Cu-MOF nanoparticles are dissolved in glutaraldehyde, and after 2.5 h, they are centrifuged, washed, and then dissolved in PBS. The capture antibody is added, and the mixture is incubated for 12 h. After centrifugation to remove excess antibody, the mixture is blocked with BSA for 1 h to avoid non-specific adsorption. After centrifugation and washing again, the mixture is dissolved in PBS and stored at 4 °C for later use.

[0015] Furthermore, in step (2), the Cu-MOF nanoparticles are uniform spherical nanoparticles, with Cu, N, O, and C elements uniformly distributed in the Cu-MOF nanoparticles, an average diameter of 115 nm, and abundant amino groups. The abundant amino groups on the surface of the Cu-MOF nanoparticles make it easy to modify the biomarker antibody to be detected, and the reaction can be carried out in an aqueous phase at room temperature.

[0016] Furthermore, in step (2), the preparation of azide-treated Escherichia coli is as follows: deionized water, buffer, restriction endonuclease, pET-28a(+) plasmid and pEGFP-N3 plasmid are mixed in an EP tube, digested in an incubator for 1 h, then Tris buffer, pET-28a(+), EGFP and T4 DNA ligase are added, incubated at 22℃ for 20 min, and then stored at -20℃;

[0017] Before transferring the recombinant plasmid, competent Escherichia coli needs to be prepared: dilute the bacteria to a suitable OD value, centrifuge after ice bath, remove the supernatant, add pre-cooled calcium chloride to the precipitate for resuspending, incubate on ice for 20 min again, centrifuge and discard the supernatant, add pre-cooled calcium chloride for resuspending.

[0018] Add the PET28a-EGFP plasmid to the above competent BL-21(DE3) E. coli, mix gently, incubate on ice for 20 min, then heat at 42°C for 65 s, and immediately place on ice and heat for 3 min. Add Kana (200 μL) to LB agar plates, place on the plates for 40 min, and incubate in an inverted position overnight at 37°C.

[0019] Single colonies were picked from the culture medium and inoculated into LB medium containing kana. The culture was incubated at 37°C for 12 hours. The bacterial solution was diluted to an OD value of 0.4-0.5, and IPTG was added. After incubation for 4 hours, EGFP-expressing E. coli were obtained.

[0020] The bacterial suspension containing the recombinant plasmid was adjusted to an OD value of 0.4-0.6, KDO-N3 was added, and after incubation for 10 h, the suspension was washed three times with PBS and centrifuged to remove unbound azide groups, thus obtaining azide-treated Escherichia coli.

[0021] The preparation method of the pET-28a-EGFP recombinant plasmid of the present invention includes the following steps: (1) Add 9 μL of ultrapure water, 3 μL of Tris buffer (containing 0.5M pH 8.0 Tris-HCl, 0.1M MgCl2, 0.5M NaCl), 1 μL of Not I restriction endonuclease, and 2 μL of BamHI restriction endonuclease to two 0.2 mL EP tubes respectively, and then add 15 μL of pET-28a(+) plasmid (referred to as tube 1) or pEGFP-N3 plasmid (referred to as tube 2) respectively; (2) Mix the two EP tubes and centrifuge them briefly, and place them in a constant temperature incubator at 37℃ for 1 hour for enzyme digestion reaction; (3) Then, add 2.5 μL of Tris buffer, 8 μL of enzyme-digested pET-28a(+), 14 μL of enzyme-digested EGFP and 3 μL of not I restriction endonuclease to the 0.2 mL EP tubes respectively. After mixing with T4 DNA ligase and briefly centrifuging, the recombinant expression vector pET-28a-EGFP was obtained by incubating at 22°C for 20 minutes. It was then stored at -20°C for transformation.

[0022] The recombinant plasmid pET28a-EGFP was used as a vector. The 5' sequencing primers and sequences were: T7: 5'-TAATACGACTCACTATAGGG-3'; and the 3' sequencing primers and sequences were: T7t: 5'-GCTAGTTATTGCTCAGCGG-3'.

[0023] The recombinant plasmid pET28a-EGFP was tagged with EGFP. The 5' sequencing primers and sequences were: CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3'; EGFP-N: 5'-CGTCGCCGTCCAGCTCGACCAG-3'.

[0024] Beneficial effects: This invention has strong detection specificity, good stability and accuracy, and a detection limit lower than the clinical gold standard ELISA. It allows for visual detection of biomarker concentrations, can be applied to stable clinical testing, and has a wider detection range.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) This invention provides an ultrasensitive fluorescence counting immunoassay method (referred to as FBLISA). This method uses a cascaded signal amplification strategy to catalyze a bioorthogonal reaction for the fixation and proliferation of fluorescent bacteria by degrading nano-copper-based metal-organic frameworks (Cu-MOF). The bioorthogonal reaction is a signal conversion method that combines the high selectivity and specificity of immunoassay with high efficiency. Fluorescent bacteria are a novel type of signal reporter bacteria with unique fluorescence characteristics, high light stability, and low background, and can be directly counted using a conventional fluorescence microscope.

[0027] (2) Due to the excellent self-replication ability of *E. coli*, this invention effectively amplifies the fluorescence signal by controlling the incubation time to achieve the sensitivity required for low-concentration detection. Verification showed that the limits of detection (LODs) for carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA) were 74.1 fg / mL and 7.2 fg / mL, respectively, which is 1000 times more sensitive than conventional ELISA. Encouragingly, FBLISA has been successfully applied to the detection of PSA in real serum samples from prostate cancer patients, demonstrating the great potential of this method in the clinical diagnosis of cancer.

[0028] (3) This method uses a cascaded signal amplification strategy to catalyze the click reaction of fluorescent bacteria fixation by degrading nano-copper-based metal-organic frameworks (Cu-MOF) (a signal conversion method with high selectivity, specificity and high efficiency). It utilizes the advantages of fluorescent bacteria such as fluorescence characteristics, high light stability and low background to directly count and detect biomarkers using a conventional fluorescence microscope.

[0029] (4) Due to the excellent self-replication ability of *E. coli*, this invention effectively amplifies the fluorescence signal by controlling the incubation time to achieve the sensitivity required for low-concentration detection. This detection method converts protein concentration into visual fluorescence counting, reducing instrument requirements; a common fluorescence microscope can be used for counting. Verification showed that the limits of detection (LODs) for carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA) were 74.1 fg / mL and 7.2 fg / mL, respectively, which is 1000 times more sensitive than conventional ELISA. Furthermore, FBLISA has been successfully applied to the detection of PSA in real serum samples from prostate cancer patients, demonstrating the great potential of this method in the clinical diagnosis of cancer.

[0030] (5) D-amino acids have been found to specifically bind to bacterial peptidoglycans and proteins (which have chemical groups required for bioorthogonal reactions) and can be used for bacterial labeling. Utilizing bioorthogonal chemical groups in the Cu(I)-catalyzed azide / alkyne cycloaddition CuAAC reaction will provide a potential transformative strategy for fluorescent biosensing based on fluorescent bacteria. Attached Figure Description

[0031] Figure 1 Characterization diagrams of the Cu-MOF prepared in this invention are shown below;

[0032] Figure 2 This is a graph showing the protein quantification analysis based on the FBLISA system of this invention;

[0033] Figure 3 This is a graph showing the specificity of the FBLISA for detecting CEA and PSA in this invention;

[0034] Figure 4 This is a graph showing the detection and analysis of clinical serum samples using the FBLISA method of this invention. Detailed Implementation

[0035] The present invention will be further described below with reference to embodiments and accompanying drawings, but this does not limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0036] Example 1

[0037] The present invention discloses a method for detecting bacteria fixed by bioorthogonal reaction in an ultrasensitive fluorescence counting immunoassay, comprising the following steps:

[0038] (1) pET-28a-EGFP recombinant plasmid was transformed into E. coli and IPTG was used to induce E. coli to express green fluorescent protein;

[0039] (2) Cu-MOF nanoparticles were dissociated with ascorbic acid to reduce Cu(II) to Cu(I); KDO-N3 (3-deoxy-D-manno-2-octulose azidosaccharide) was used to specifically incorporate the azido group into the lipopolysaccharide on the E. coli membrane, thus functionalizing it with azidosaccharide; the azido group of the azido-modified E. coli reacted with the alkyne group attached to the BSA coated on the 96-well plate to fix the azido-modified E. coli on the plate; bovine serum albumin and N-propargyl maleimide were mixed in PBS and concentrated by ultrafiltration to obtain alkyne-modified bovine serum albumin BSA, with a mass ratio of alkyne-modified bovine serum albumin BSA to N-propargyl maleimide of 25:1. Cu-MOF was prepared by a high-temperature and high-pressure reaction using copper nitrate trihydrate: PVP was dissolved in a mixed solvent of DMF and ethanol to prepare solution A; then, copper nitrate trihydrate and 2-aminoterephthalic acid were dissolved in DMF to synthesize solution B. Solutions A and B were mixed and reacted in a high-pressure reactor at 100°C for 12 hours to obtain Cu-MOF. Antibodies capturing the cancer-specific antigen CEA were then modified onto Cu-MOF nanoparticles. The Cu-MOF nanoparticles were first dissolved in glutaraldehyde for 2.5 hours, then centrifuged, washed, and dissolved in PBS. The capturing antibody was added, and the mixture was incubated for 12 hours. After centrifugation to remove excess antibody, the nanoparticles were blocked with BSA for 1 hour to prevent non-specific adsorption. After another centrifugation and washing, the nanoparticles were dissolved in PBS and stored at 4°C for later use. The Cu-MOF nanoparticles are uniform spherical nanoparticles with Cu, N, O, and C elements uniformly distributed within them. The average diameter is 115 nm, and they contain abundant amino groups. The abundant amino groups on the surface of the Cu-MOF nanoparticles make it easy to modify the biomarker antibodies to be detected, and the reaction can be carried out in an aqueous phase at room temperature.

[0040] Preparation of azide-treated Escherichia coli: Deionized water, buffer, restriction endonuclease, pET-28a(+) plasmid and pEGFP-N3 plasmid were mixed in an EP tube and digested in an incubator for 1 h. Tris buffer, pET-28a(+), EGFP and T4 DNA ligase were added and mixed. The mixture was incubated at 22 °C for 20 min and then stored at -20 °C.

[0041] Before transferring the recombinant plasmid, competent Escherichia coli needs to be prepared: dilute the bacteria to a suitable OD value, centrifuge after ice bath, remove the supernatant, add pre-cooled calcium chloride to the precipitate for resuspending, incubate on ice for 20 min again, centrifuge and discard the supernatant, add pre-cooled calcium chloride for resuspending.

[0042] Add the PET28a-EGFP plasmid to the above competent BL-21(DE3) E. coli, mix gently, incubate on ice for 20 min, then heat at 42°C for 65 s, and immediately place on ice and heat for 3 min. Add Kana (200 μL) to LB agar plates, place on the plates for 40 min, and incubate in an inverted position overnight at 37°C.

[0043] Single colonies were picked from the culture medium and inoculated into LB medium containing kana. The culture was incubated at 37°C for 12 hours. The bacterial solution was diluted to an OD value of 0.4-0.5, and IPTG was added. After incubation for 4 hours, EGFP-expressing E. coli were obtained.

[0044] The bacterial suspension containing the recombinant plasmid was adjusted to an OD value of 0.4-0.6, KDO-N3 was added, and after incubation for 10 h, the suspension was washed three times with PBS and centrifuged to remove unbound azide groups, thus obtaining azide-treated Escherichia coli.

[0045] The present invention relates to a set of pET-28a-EGFP recombinant plasmids that can express EGFP fluorescence.

[0046] (3) The pET-28a-EGFP recombinant plasmid was transformed into Escherichia coli and induced to be transcribed into the T promoter by IPTG. This induced E. coli to express green fluorescent protein, converting the concentration of the biomarker into fluorescent bright spots of fluorescent E. coli, thereby achieving quantitative detection. The signal amplification effect was achieved by the proliferation of azidated E. coli, and the signal amplification time was 20-30 min.

[0047] The preparation method of the pET-28a-EGFP recombinant plasmid of the present invention includes the following steps: (1) Add 9 μL of ultrapure water, 3 μL of Tris buffer (containing 0.5M pH8.0 Tris-HCl, 0.1M MgCl2, 0.5M NaCl), 1 μL of Not I restriction endonuclease, and 2 μL of BamHI restriction endonuclease to two 0.2 mL EP tubes respectively, and then add 15 μL of pET-28a(+) plasmid (referred to as tube 1) or pEGFP-N3 plasmid (referred to as tube 2) respectively; (2) Mix the two EP tubes and centrifuge them briefly, and place them in a constant temperature incubator at 37℃ for 1 hour for enzyme digestion reaction; (3) Then, add 2.5 μL of Tris buffer, 8 μL of enzyme-digested pET-28a(+), 14 μL of enzyme-digested EGFP and 3 μL of not I restriction endonuclease to the 0.2 mL EP tubes respectively. After mixing with T4 DNA ligase and briefly centrifuging, the recombinant expression vector pET-28a-EGFP was obtained by incubating at 22°C for 20 minutes. It was then stored at -20°C for transformation.

[0048] Example 2

[0049] When E. coli expressing EGFP was induced by IPTG and the recombinant plasmid was introduced, the bacterial reproduction rate was the fastest, the signal amplification effect was the strongest, and the sensitivity was the highest when the volume ratio of LB medium to PBS was 9:1.

[0050] Example 3

[0051] Experiments have verified that only Cu(I) can catalyze the target bioorthogonal reaction, while other metal ions did not show any signal. Furthermore, under the same conditions, sodium citrate, glutathione, and ascorbic acid, as reducing agents, can all catalyze the production of Cu(I) from CuCl2 and Cu-MOF, but AA exhibits the highest catalytic efficiency. Moreover, the amount of Cu-MOF is directly proportional to the number of immobilized fluorescent bacteria, confirming the validity of the immunoassay mechanism established in this invention.

[0052] Example 4

[0053] Validation of bioorthogonal reactions mediated by metal-organic framework nanoparticles

[0054] DBCO-Cy5 was labeled onto azide-modified *E. coli* via a click reaction. EGFP-labeled *E. coli* exhibited significant red fluorescence, indicating successful azide modification. AA was added to degrade Cu-MOF. As designed, Cu(II) was reduced to Cu(I), and the azide group reacted with the alkyne group attached to BSA coated on a 96-well plate via a click reaction, immobilizing *E. coli* on the 96-well plate. This invention uses alkyne-modified BSA to draw different patterns on the plate; after binding, *E. coli* expresses fluorescence at the drawn patterns. This system can immobilize *E. coli* on a plate.

[0055] Example 5

[0056] Sensitivity of FBLISA

[0057] Carcinoembryonic antigen (CEA) is a broad-spectrum tumor marker that can reflect the presence of various tumors. It is a tumor marker for assessing the treatment efficacy, disease progression, and prognosis of colorectal cancer, breast cancer, and lung cancer.

[0058] Prostate-specific antigen (PSA) is an important protein signal for prostate cancer and is the most effective biomarker for detecting prostate cancer.

[0059] Gradually increasing the concentrations of CEA and PSA in the samples resulted in an increase in the number of fluorescent bacteria, with CEA ranging from 0.001 to 100 ng / mL and PSA from 0.0001 to 100 ng / mL. CEA and PSA showed linear trends in the ranges of 0.001–0.1 ng / mL and 0.0001–0.005 ng / mL, respectively.

[0060] The LOD was calculated based on the linear slope of three times the standard deviation of the blank signal (n=3). The LODs for CEA and PSA were 0.7 pg / mL and 63.1 fg / mL, respectively, which are lower than the detection limits of the gold standard ELISA method.

[0061] Escherichia coli can multiply every 20-30 minutes. After 1 hour of culture, the signal is amplified due to its proliferation. The metabolic growth curve of fluorescent E. coli shows that the bacteria grows fastest within 1 hour. After culturing E. coli at 37℃ for 1 hour, the LOD values ​​of CEA and PSA decreased to 74.1 fg / mL and 7.2 fg / mL, respectively. The linear dynamic range of FBLISA for detecting CEA and PSA concentrations increased from 0.0001 to 0.1 ng / mL and 0.00001 to 0.005 ng / mL, respectively, both an order of magnitude wider than before signal amplification.

[0062] The minimum detectable concentration of PSA is 7.2 fg / mL, approximately 1000 times lower than that of traditional ELISA kits (0.01 ng / mL). Notably, compared to traditional ELISA, PSA has a 10-fold expanded detection range, making it more suitable for directly detecting high-concentration samples (>10 ng / mL) without requiring the dilution methods of ELISA. Figure 2 ).

[0063] FBLISA has great potential for application in practical clinical diagnosis and protein quantification analysis in many fields such as biological systems and precision medicine.

[0064] Example 6

[0065] To verify the feasibility of the above scheme, the morphology of Cu-MOF was characterized using scanning electron microscopy (SEM, S-3400, Hitachi) and transmission electron microscopy (TEM, Jem2010, Jeol). The Zeta potential of Cu-MOF was measured using a Zeta sizer nano ZS instrument (Morven).

[0066] The recombinant plasmid pET28a-EGFP was used as a vector. The 5' sequencing primers and sequences were: T7: 5'-TAATACGACTCACTATAGGG-3'; and the 3' sequencing primers and sequences were: T7t: 5'-GCTAGTTATTGCTCAGCGG-3'.

[0067] The recombinant plasmid pET28a-EGFP uses EGFP as a tag. The 5' sequencing primers and sequences are: CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3'; EGFP-N: 5'-CGTCGCCGTCCAGCTCGACCAG-3'.

[0068] Deionized water, buffer, restriction endonuclease, pET-28a(+) plasmid and pEGFP-N3 plasmid were mixed in an EP tube and digested in an incubator for 1 h. Tris buffer, pET-28a(+), EGFP and T4 DNA ligase were added and mixed. The mixture was incubated at 22 °C for 20 min and then stored at -20 °C.

[0069] Before transferring the recombinant plasmid, competent E. coli need to be prepared: dilute the bacteria to a suitable OD value, centrifuge after ice bath, remove the supernatant, add pre-cooled calcium chloride to the precipitate for resuspending, incubate on ice again for 20 min, centrifuge and discard the supernatant, add pre-cooled calcium chloride for resuspending.

[0070] Add the PET28a-EGFP plasmid to the competent BL-21(DE3) E. coli cells, mix gently, incubate on ice for 20 min, then heat at 42°C for 65 s, and immediately place on ice and heat for 3 min. Add 200 μL of Kana to LB agar plates, place on the plates for 40 min, and incubate in an inverted manner overnight at 37°C.

[0071] Single colonies were picked from the culture medium and inoculated into LB medium containing kana. The culture was carried out at 37°C for 12 hours. The bacterial solution was diluted to an OD value of 0.4-0.5 and then IPTG was added. After culturing for 4 hours, EGFP-expressing E. coli were obtained.

[0072] The bacterial suspension containing the recombinant plasmid was adjusted to an OD value of 0.4-0.6, KDO-N3 was added, and after incubation for 10 h, the suspension was washed three times with PBS and centrifuged to remove unbound azide groups, thus obtaining azide-treated Escherichia coli.

[0073] Bovine serum albumin and N-propargyl maleimide were mixed in PBS, shaken overnight for 24 hours, centrifuged to remove unbound N-propargyl maleimide, concentrated by ultrafiltration and washed three times, and stored at 4°C for later use.

[0074] Example 7

[0075] FBLISA Specificity

[0076] To evaluate the specificity of FBLISA, other proteins at concentrations ten times higher than the analyte were introduced. The signal induced by the analyte could be clearly separated from the other proteins, demonstrating that the detection platform of this invention can be used for the simultaneous detection of the analyte and other proteins. These results indicate that FBLISA can specifically detect the analyte because the antibody has a good specific affinity for the analyte and is not affected by interference from other proteins. Figure 3 ).

[0077] Example 8

[0078] Application of FBLISA in Clinical Sample Analysis

[0079] PSA levels in 30 clinical serum samples (15 prostate cancer patients and 15 healthy adults) diagnosed using the clinical gold standard method were measured using FBLISA. The FBLISA results were compared with those of the traditional HRP-ELISA method, and a receiver operating characteristic (ROC) curve for PSA detection was constructed: the area under the curve (AUC) was 0.938 (95% CI: 0.846-1, ****P<0.0001, highly significant), the optimal cutoff value was 4.115 ng / mL, the clinical sensitivity was 86.7%, and the specificity was 97%. These results indicate that FBLISA has relatively high accuracy. Interclass correlation coefficient (ICC) and Bland-Altman analysis were used to assess the concordance between FBLISA and ELISA. The ICC for PSA detection was 0.933, and most results for Bland-Altman detection were within the 1.96 SD range. Therefore, FBLISA and ELISA are highly concordant. Meanwhile, FBLISA and ELISA results showed a very strong correlation (r = 0.916). A clinical threshold (4.0 ng / mL) was used to distinguish between positive and negative samples. Figure 4 The red horizontal dashed line indicates that there is a high degree of consistency between the actual and expected values, demonstrating FBLISA's ability to analyze clinical samples.

[0080] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope. The scope of protection of this invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A method for detecting bacteria immobilized by a bioorthogonal reaction using ultrasensitive fluorescence counting immunoassay, characterized in that... Includes the following steps: (1) The pET-28a-EGFP recombinant plasmid was transformed into E. coli, and the expression of green fluorescent protein in E. coli was induced by IPTG; (2) Cu-MOF nanoparticles were dissociated with ascorbic acid to reduce Cu(II) to Cu(I). The azide group modified by azido-modified Escherichia coli was combined with the alkynyl group on the alkynylated bovine serum albumin. The azide group and the alkynyl group under the catalysis of Cu(I) underwent a click reaction to achieve the effect of immobilizing azido-modified Escherichia coli on a 96-well plate. Cu-MOF was obtained by reacting copper nitrate trihydrate with high temperature and pressure: PVP was dissolved in a mixed solvent of DMF and ethanol to prepare solution A; copper nitrate trihydrate and 2-aminoterephthalic acid were dissolved in DMF to prepare solution B. Solutions A and B were mixed and reacted in a high-pressure reactor at 100°C for 12 hours to obtain Cu-MOF. Antibodies that capture cancer-specific antigen CEA were modified onto Cu-MOF nanoparticles. Cu-MOF nanoparticles were first dissolved in glutaraldehyde, centrifuged and washed for 2.5 hours, then dissolved in PBS, and the capture antibody was added. After incubation for 12 hours, after centrifugation to remove excess antibody, BSA was blocked for 1 hour to avoid non-specific adsorption. After centrifugation and washing again, the nanoparticles were dissolved in PBS and stored at 4°C for later use. (3) The concentration of biomarkers is converted into fluorescent bright spots of fluorescent Escherichia coli by the self-replication of luminescent Escherichia coli, thereby realizing quantitative detection. The signal amplification effect is achieved by the proliferation of azidated Escherichia coli, and the signal amplification time is 20-30 min.

2. The detection method for ultrasensitive fluorescence counting immunoassay of bacteria fixed by bioorthogonal reaction according to claim 1, characterized in that: In step (2), bovine serum albumin and N-propyne maleimide are mixed in PBS and concentrated by ultrafiltration to obtain alkynylated bovine serum albumin. The mass ratio of alkynylated bovine serum albumin to N-propyne maleimide is 25:

1.

3. The detection method for ultrasensitive fluorescence counting immunoassay of bacteria fixed by bioorthogonal reaction according to claim 1, characterized in that: In step (2), KDO-N3 (3-deoxy-D-manno-2-octulose azide) specifically incorporates the azide group into the lipopolysaccharide on the E. coli membrane, thereby functionalizing it with azide; the azide group reacts with the alkyne group attached to the BSA coated on the 96-well plate, and the E. coli is immobilized on the 96-well plate.

4. The detection method for ultrasensitive fluorescence counting immunoassay of bacteria fixed by bioorthogonal reaction according to claim 1, characterized in that: In step (2), the preparation of azide-treated Escherichia coli: deionized water, buffer, restriction endonuclease, pET-28a(+) plasmid and pEGFP-N3 plasmid were mixed in an EP tube, digested in an incubator for 1 h, and then Tris buffer, pET-28a(+), EGFP and T4 DNA ligase were added. After incubation at 22℃ for 20 min, the mixture was stored at -20℃. Before transferring the recombinant plasmid, competent Escherichia coli needs to be prepared: dilute the bacteria to a suitable OD value, centrifuge after ice bath, remove the supernatant, add pre-cooled calcium chloride to the precipitate for resuspending, incubate on ice for 20 min again, centrifuge and discard the supernatant, add pre-cooled calcium chloride for resuspending. Add the PET28a-EGFP plasmid to the competent E. coli cells, mix gently, incubate on ice for 20 min, heat at 42°C for 65 s, and immediately place on ice and heat for 3 min; add 200 μL of Kana to an LB agar plate, place on the plate for 40 min, and incubate upside down in a 37°C incubator overnight. Single colonies were picked from the culture medium and inoculated into LB medium containing kana. The culture was incubated at 37°C for 12 hours. The bacterial solution was diluted to an OD value of 0.4-0.5, and IPTG was added. After incubation for 4 hours, EGFP-expressing E. coli were obtained. The bacterial suspension containing the recombinant plasmid was adjusted to an OD value of 0.4-0.6, KDO-N3 was added, and after incubation for 10 h, the suspension was washed three times with PBS and centrifuged to remove unbound azide groups, thus obtaining azide-treated Escherichia coli.

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