Application of manganese-doped zinc germanate nanorod fluorescent probe in anti-hypochlorous acid and sodium hypochlorite interference aspect of blood analysis

Abstract

The invention discloses application of a manganese-doped zinc germanate nanorod fluorescent probe in hypochlorous acid and sodium hypochlorite interference resistance in blood analysis, relates to the technical field of germanium-containing luminescent materials, and aims to solve the problem that hypochlorous acid and sodium hypochlorite can interfere blood detection. After the photobleached manganese-doped zinc germanate nanorod fluorescent probe disclosed by the invention is in contact with a sample containing hemoglobin, hypochlorous acid or sodium hypochlorite, fluorescence and continuous luminescence properties are recovered; the attenuation rate after the hypochlorous acid or sodium hypochlorite induced fluorescence and continuous luminescence are recovered is greater than the attenuation rate after the hemoglobin induced fluorescence and continuous luminescence are recovered; according to the method, residual bloodstains which are cleaned by hypochlorous acid or sodium hypochlorite and cannot be seen by naked eyes in the sun can be identified and detected by utilizing the characteristic that the fluorescence decay rate after the reaction fluorescence of photobleached ZGO: Mn NRs and hypochlorous acid or sodium hypochlorite is recovered is greater than the fluorescence decay rate after the fluorescence induced by hemoglobin is recovered.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence analysis of germanium-containing luminescent materials, and specifically to the application of manganese-doped zinc germanate nanorod fluorescent probes in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis. Background Art

[0002] Photobleaching refers to the phenomenon that fluorophores lose or permanently reduce their luminescence ability after long-term exposure to light. This phenomenon is usually not conducive to the application of long-afterglow luminescent materials. Photobleaching is widely regarded as a key limitation in optical sensing and bioimaging because it has an adverse impact on detection accuracy, sensitivity, and reproducibility. Therefore, various means have been adopted to improve the photostability of fluorophores. However, some studies have shown that this property of photobleaching can be utilized.

[0003] The invention patent with the publication number CN116515483B and the name of a novel fluorescent "turn-on" type fluorescent probe and its application in detecting copper ions in lake water discloses a manganese-doped zinc germanate nanorod fluorescent probe and its preparation method, and records the related application of detecting copper ions. In fact, it utilizes the fluorescence recovery after photobleaching of this material, which also broadens the idea for further research and utilization of the photobleaching property.

[0004] Criminal investigation blood detection is an important task in forensic investigations. Current HGB detections rely on catalytic oxidation assays using commercial fluorescein-based PL or luminol-based chemiluminescence (CL) reagents. Although these methods are effective for bloodstain visualization, they have inherent limitations; common bleaching agents generally contain strong oxidants such as hypochlorous acid and sodium hypochlorite, which can react with luminol and emit strong fluorescence, thus masking the presence of bloodstains. Especially in the case where the crime scene has been thoroughly treated with a bleaching agent, the detection effect of luminol will be greatly reduced.

[0005] The invention patent with the publication number CN111610168B and the name of an AIE molecule for detecting potential bloodstains and its application not only records that the luminescence time of luminol is extremely short, which is not conducive to forensic investigators extracting bloodstain morphology evidence, but also records that fluorescein derivatives are subject to rapid photobleaching; this patent discloses that tetraphenylethylene maleimide (TPE-MI) undergoes a click reaction with the thiol groups in serum albumin under mild conditions, and the molecule emits cyan fluorescence under ultraviolet lamp excitation due to restricted movement in the protein cavity after the reaction. Although this method can achieve longer luminescence after contacting blood, it cannot solve the above problems of interference from hypochlorous acid and sodium hypochlorite. Summary of the Invention

[0006] The object of the present invention is to provide the application of manganese-doped zinc germanate nanorod fluorescent probes in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis, so as to solve the problem that hypochlorous acid and sodium hypochlorite will interfere with blood detection.

[0007] To achieve the above object, the present invention provides the following technical solution: the application of manganese-doped zinc germanate nanorod fluorescent probes in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis. After the photo-bleached manganese-doped zinc germanate nanorod fluorescent probes come into contact with a sample containing hemoglobin, they restore fluorescence and persistent luminescence properties, and hemoglobin can prevent the re-photo-bleaching of the manganese-doped zinc germanate nanorod fluorescent probes; after the photo-bleached manganese-doped zinc germanate nanorod fluorescent probes come into contact with a sample containing hypochlorous acid or sodium hypochlorite, they restore fluorescence and persistent luminescence properties, but the decay rate of fluorescence and persistent luminescence over time is greater than the decay rate after the fluorescence and persistent luminescence are restored induced by hemoglobin.

[0008] Preferably, the above sample containing hemoglobin includes a sample mixed with hypochlorous acid or sodium hypochlorite.

[0009] Preferably, the above sample containing hemoglobin can be undiluted real blood or diluted blood.

[0010] Preferably, the above sample containing hemoglobin can be fresh or old, can be visible blood stains, or can be latent blood stains invisible under sunlight.

[0011] Preferably, the above sample containing hemoglobin can be from different material surfaces.

[0012] Preferably, photo-bleaching is ultraviolet irradiation at 100-365 nm for 1-30 min.

[0013] Preferably, the preparation method of the manganese-doped zinc germanate nanorod fluorescent probes includes: mixing Zn(NO3)2, Mn(NO3)2 and concentrated nitric acid, adding them to deionized water and stirring vigorously, then adding a Na2GeO3 solution, quickly adding ammonium hydroxide to adjust the pH of the system to 6-10 while stirring vigorously, then transferring the reaction solution to a reaction kettle and continuing to stir at room temperature for 1-24 h, and then placing the reaction kettle at 100-300 °C for reaction for 4-24 h, and centrifuging and washing the product with deionized water to obtain ZGO:Mn NRs.

[0014] Another technical solution provided by the present invention: the application of manganese-doped zinc germanate nanorod fluorescent probes in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis. After the manganese-doped zinc germanate nanorod fluorescent probes are photo-bleached, they are used for detecting invisible residual blood stains under sunlight after being washed with hypochlorous acid or sodium hypochlorite at the crime scene.

[0015] Preferably, the above detection method is to cover the area to be detected with the manganese-doped zinc germanate nanorod fluorescence probe solution after photobleaching, continuously irradiate with the excitation light, and observe whether the fluorescence intensity is higher than that of the surrounding area after the fluorescence decays over time, or turn off the excitation light after irradiating with the excitation light, and collect and observe whether the persistent luminescence intensity is higher than that of the surrounding area after the persistent afterglow decays.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] 1. The application of the manganese-doped zinc germanate nanorod fluorescence probe in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis can utilize the fluorescence recovery after the reaction of ZGO:Mn NRs after photobleaching with hemoglobin to detect bloodstain samples containing hemoglobin. It can also utilize the fluorescence decay rate of ZGO:Mn NRs after the reaction with hypochlorous acid or sodium hypochlorite and fluorescence recovery being much greater than the fluorescence decay rate after the fluorescence recovery induced by hemoglobin to identify and detect invisible residual bloodstains under sunlight after being washed with hypochlorous acid or sodium hypochlorite. The detection method is simple, only need to quench the fluorescence of the probe and then cover it on the area to be detected. Moreover, ZGO:Mn NRs after photobleaching have high sensitivity to hemoglobin, low detection limit, the recognition is not limited to the water environment or different material surfaces, and it has strong selectivity and anti-interference ability to hemoglobin, which can effectively avoid the influence of common components in blood.

[0018] 2. The application of the manganese-doped zinc germanate nanorod fluorescence probe in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis can, in addition to observing the fluorescence characteristics under the excitation light after the reaction of ZGO:Mn NRs after photobleaching with hemoglobin, also collect and observe the persistent afterglow by turning off the excitation light. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the fluorescence emission decay curve of ZGO:Mn NRs prepared under various pH conditions in Example 1 after irradiation with a 254 nm ultraviolet lamp;

[0020] Figure 2 is Figure 1 the fluorescence photobleaching photo of ZGO:Mn NRs prepared under the pH 9.5 condition in

[0021] Figure 3 is Figure 1 the fluorescence photobleaching photo of ZGO:Mn NRs prepared under the pH 8 condition in

[0022] Figure 4 It is the fluorescence spectrum ( Figure 4 a) and the persistent luminescence spectrum ( Figure 4c); and fluorescence at 536 nm ( Figure 4 b) and persistent luminescence ( Figure 4 d) Curve of the intensity versus the added HGB concentration and the corresponding linear graph;

[0023] Figure 5 Scatter plot of the photostability of the ZGO:Mn NRs solution over time after adding HGB in Example 3, Figure 5 a is fluorescence, Figure 5 b is persistent luminescence;

[0024] Figure 6 a is the decrease in the photobleached fluorescence intensity of ZGO:Mn NRs before and after irradiation in deionized water and DO water; Figure 6 b and Figure 6 c is the EPR spectrum of ZGO:Mn NRs for detecting ROS before and after irradiation; Figure 6 d is the curve of the photobleaching behavior of ZGO:Mn NRs after adding a ROS scavenger;

[0025] Figure 7 Reaction kinetic curve after adding 12 μM HGB to ZGO:Mn NRs prepared under the condition of pH 9.5 in Example 1, diluted to 0.12 mg / ml and photobleached ( Figure 7 a), Zeta potential of ZGO:Mn NRs ( Figure 7 b); Figure 7 c is the EPR spectrum of the DMPO-·O2 - signal after photobleaching and adding HGB; Figure 7 d is the ERP signal spectrum of TEPMO-h + In the figure, the black line is ZGO:Mn NRs in the dark, the green line is after irradiation for 10 minutes, and the yellow line is after adding HGB after irradiation;

[0026] Figure 8 Results of the selectivity and anti-interference test of photobleached ZGO:Mn NRs for HGB in Example 4, where Figure 8 For part b, the upper part shows the comparison of the results of 11 samples in the fluorescence group with only interference items added and the results after adding HGB to the blank sample, and the lower part shows the comparison of the results of 12 samples in the fluorescence group with HGB added on average;

[0027] Figure 9 Results of hemoglobin detection in the water environment in Example 5;

[0028] Figure 10 Fluorescence spectra of ZGO:Mn NRs before and after adding real blood in Example 5;

[0029] Figure 11Recognition of diluted blood by photo-bleached ZGO:Mn NRs in Example 5;

[0030] Figure 12 Experimental results of blood recognition on different material surfaces in Example 5;

[0031] Figure 13 Comparison of experimental results of hypochlorous acid samples with the same HGB concentration in Example 6. Specific implementation mode

[0032] Unless otherwise specified, the water used in the following examples is secondary deionized water (18.25 MΩ·cm -1 ), and the chemical raw materials used are all of analytical purity.

[0033] Example 1

[0034] Prepare a manganese-doped zinc germanate nanorod fluorescent probe, that is, Zn2GeO4 nanorods doped with Mn 2+ , hereinafter abbreviated as ZGO:Mn NRs:

[0035] The preparation process has been described in detail in the invention patent with the publication number CN116515483B and the name of "Novel Fluorescent 'Turn-on' Fluorescent Probe and Its Application in Detecting Copper Ions in Lake Water". The method in this patent can be directly used for preparation: Mix Zn(NO3)2, Mn(NO3)2 and concentrated nitric acid, add them to deionized water and stir vigorously. Subsequently, add Na2GeO3 solution, and quickly add ammonia water to adjust the pH of the system to 6 - 8 while stirring vigorously. Then transfer the above reaction solution to a reaction kettle and continue to stir at room temperature for 1 - 24 h, and then place the reaction kettle at 100 - 300 °C for reaction for 4 - 24 h. Centrifuge and wash the product with deionized water and then dry it to obtain ZGO:Mn NRs, and then perform alkalization treatment.

[0036] Or optimize to a certain extent. The alkalization treatment step of ZGO:Mn NRs can be omitted. This step aims to increase the fluorescence quenching efficiency, which is not necessary, but it can indeed reduce the fluorescence intensity more. Correspondingly, this performance can be improved by adjusting the pH during the preparation process, thus simplifying the preparation process. In the following, this example uses an improved hydrothermal method for synthesis.

[0037] First, 2.08 g of GeO2 was dissolved in 50 mL of 2 mol / L NaOH to prepare a Na2GeO3 solution with a concentration of 0.4 mol / L. Secondly, 2 mL of 1 mol / L Zn(NO3)2, 1 mL of 5 mmol / L Mn(NO3)2 and 300 μL of concentrated HNO3 were added to 8 mL of deionized water. Then, under vigorous stirring, 2.5 mL of 0.4 mol / L Na2GeO3 was slowly added to the above solution, and a white precipitate was instantly formed in the system. Subsequently, ammonium hydroxide (weight percentage 28%) was immediately added to adjust the pH value of the solution. The resulting reaction system was stirred and allowed to stand at room temperature for 1 hour. Then the solution was transferred to a 25 mL Teflon-lined autoclave and reacted continuously at 220 °C for 4 hours. After the reaction, the solid sample was collected by centrifugation (8000 r / min), then washed three times with pure water and centrifuged (10000 r / min). Finally, the collected ZGO:Mn NRs were dispersed in deionized water to prepare a solution with a concentration of 0.12 g / mL and stored in a refrigerator at 2-8 °C for later use.

[0038] The pH meter used in this example is Mettler Toledo FE28-Bio, and the following analyses are all based on the test results of this device:

[0039] During the preparation process, adding ammonium hydroxide to adjust the pH value of the solution can be 6-10. In this example, five products were prepared under five conditions of 7, 8, 9, 9.5, and 10. ZGO:Mn NRs prepared under different pH conditions were respectively configured into 0.12 mg / mL samples with deionized water for experiments. A fluorescence spectrometer (Hitachi F-7100) was used to synchronously collect the fluorescence and persistent luminescence intensity data of ZGO:Mn NRs under different irradiation times. The fluorescence parameters were set with an excitation wavelength of 254 nm, an emission wavelength of 536 nm, both the incident slit and the emission slit were set to 5 nm, and the PMT voltage was set to 570 V. The persistent luminescence parameters were set with an excitation wavelength of 254 nm, an emission wavelength of 530 nm, the incident slit was set to 20 nm, the emission slit was set to 5 nm, and the PMT voltage was set to 570 V; Figure 1 This is the fluorescence decay curve of each sample after ultraviolet irradiation at 254 nm, and its persistent luminescence decay curve has the same trend as the fluorescence decay curve.

[0040] From Figure 1 it can be seen that it is recommended to add ammonium hydroxide to adjust the pH value of the solution during the preparation process to be 8-10, with 9.5 being the best. The products prepared within the recommended and optimal pH value ranges show faster fluorescence quenching when exposed to a 254 nm ultraviolet lamp, that is, it can be understood that they are more prone to photobleaching operations. Among them, the product prepared at pH 9.5 has a fluorescence intensity reduction of 90.13% after irradiation for 590 seconds.

[0041] Due to reasons such as differences in pH meters, it is recommended to refer to this example when actually preparing and using for the first time. Figure 1 , perform preparations at various pH values, analyze the effect of pH on the photobleaching efficiency by yourself, and select the appropriate pH measured by the equipment used.

[0042] Example 2

[0043] According to the method of Example 1, a ZGO:Mn NRs solution with a concentration of 0.12 mg / mL was prepared at pH 9.5, and it was dispensed into two groups of multiple cuvettes. It was irradiated with 254 nm ultraviolet light for 600 seconds to obtain photobleached ZGO:Mn NRs as samples for standby. Two groups were respectively used for detecting HGB fluorescence and persistent luminescence experiments; Aqueous solutions of HGB with various different concentrations were prepared, and aqueous solutions of HGB were respectively added to each photobleached sample to analyze the effects at different concentrations. The specific concentrations are referred to Figure 4 a and Figure 4 the HGB concentration values in the samples after addition in c. After each addition of the HGB solution, gently mix to ensure uniform reaction, and synchronously record the fluorescence and persistent luminescence intensity signals of ZGO:Mn NRs after adding different concentrations of HGB; The relationships between fluorescence and persistent luminescence intensity and HGB concentration at different concentrations of HGB are referred to Figure 4 b and Figure 4 d;

[0044] As Figure 4 shown in a, the photobleached ZGO:Mn NRs showed significant fluorescence recovery after adding HGB. Figure 4 There is also a physical fluorescence photo under 254 nm ultraviolet light in a, and green light emission can be seen. The fluorescence linear range is 2.50 nmol·L-1–0.10 μmol·L-1, and the detection limit of hemoglobin calculated using the 3σ rule is 0.80 nmol·L-1. Figure 4 Parallel enhancement of persistent luminescence was observed in c. Figure 4 d shows a linear range from 19.80 nmol·L-1–1.20 μmol·L-1, and the limit of detection (LOD) is 3.70 nmol·L-1.

[0045] The following table is a systematic comparison of the results of this example with several current hemoglobin detection methods. Obviously, the method of this example has a wider linear range, a faster response time, and excellent detection limit levels. Importantly, compared with traditional HGB detection methods, the photobleached ZGO:Mn NRs has obvious practical advantages in cost-effectiveness, portability and user-friendliness.

[0046] Table 1 Comparison of detection limits of several probes for HGB detection with ZGO:Mn NRs

[0047]

[0048] Example 3

[0049] Take the two groups of samples added with 12 μM HGB in Example 2 and continue the long-term experiment. The experimental conditions are long-term exposure to lighting conditions (254 nm, 0.14 mW / cm 2 ) and normal contact with air. Record the fluorescence and persistent luminescence intensity vs. time graph as shown in Figure 5 . It can be seen that after more than 20 minutes, the light intensity decreases very little, and the decreasing rate gradually slows down. This finding is in sharp contrast to the luminescence of luminol for about 30 seconds. Even with illumination, the evidence that has appeared will not disappear quickly, which is very conducive to forensic investigators to extract bloodstain pattern evidence. Combining with the detection limit in Example 2, this undoubtedly confirms that the photo-bleached ZGO:Mn NRs have a bright application prospect in blood trace analysis.

[0050] In addition, after the above samples irradiated with excitation light for more than 20 minutes are placed under natural sunlight conditions for 2 days, obvious fluorescence characteristics can still be observed when irradiated with 254 nm excitation light again, and there is still persistent long afterglow luminescence characteristics after turning off the excitation light.

[0051] Investigation of the influence of ROS on photo-bleaching

[0052] In order to explore the reason for the long-term luminescence after photo-bleached ZGO:Mn NRs contact with HGB, it is necessary to re-analyze from the mechanism of photo-bleaching. In addition to the relatively conventional influences such as material concentration, light power density, and light wavelength, it is generally believed in the industry that reactive oxygen species (ROS) have a greater influence on photo-bleaching, and Figure 6 the control experiment of a also confirms this view. The fluorescence intensity of the deoxygenated aqueous solution of ZGO:Mn NRs (purged with argon) only decreased by about 17.95%, which is significantly lower than the 86.21% decrease observed in the non-deoxygenated aqueous solution; in order to determine the ROS contributor, the influence of ROS is analyzed preferentially below, and the fluorescence spectrometer parameters are set the same as in Example 1;

[0053] Use DMPO (for ·OH / ·O2 - ) and TEMP (for 1 O2) for electron paramagnetic resonance (EPR) spin trapping determination. Use the radical scavenger 2,2,6,6-tetramethylpiperidine (TEMP) to trap singlet oxygen ( 1 O2), and use 5,5-dimethyl-1-pyrrolidine N-oxide (DMPO) to trap ·OH and ·O2 -, 2,2,6,6 - tetramethylpiperidine - 1 - oxyl radical (TEMPO) was used to capture photo - generated electrons; 0.12 mg / mL of ZGO:MnNRs prepared at pH 9.5 was added to a cuvette, and then a quantitative sample was mixed with the scavenger. The radical signals were collected before illumination, after irradiation with a 254 nm ultraviolet lamp for 10 min, and after adding a low - concentration HGB 10 min after illumination. Under dark conditions, the EPR signals of ·O2−, 1 O2 and ·OH were negligible. Refer to Figure 6 b and Figure 6 c. After irradiation at 254 nm, a very small 1 O2 signal persisted, ruling out its participation. However, due to the interfacial electron transfer between hydroxyl ions (OH - ) and ZGO:Mn NRs, a strong ·OH signal appeared. To confirm the generation of ·O2−, methanol was used to replace water to inhibit competitive reactions. The EPR spectrum exhibited different DMPO - ·O2− spin - adduct characteristics, verifying the generation of ·O2−;

[0054] To further clarify the role of reactive oxygen species, 1 mmol / L TBA solution (·OH scavenger), 1 mmol / L NaN3 solution ( 1 O2 scavenger), and 10 μg / mL SOD solution (·O2 - scavenger) were prepared respectively. First, using the TBA solution as the solvent, ZGO:Mn NRs prepared under the conditions of pH 9.5 in Example 1 were added to a cuvette to a concentration of 0.12 mg / mL. Subsequently, the cuvette was placed under an ultraviolet lamp (254 nm) for irradiation treatment, and a fluorometer was used to synchronously collect the fluorescence intensity of ZGO:MnNRs at different irradiation times to explore the effect of ·OH on the fluorescence of NRs. The operations for the other two solvents were the same, and the effects of ·O2 - , 1 O2 on the fluorescence of NRs were explored respectively. When TBA and NaN3 were added, the photobleaching behavior changed little, indicating that 1 O2 and ·OH were not the main reasons for the photobleaching of ZGO:Mn NRs during irradiation. However, after adding SOD, the recovery of the fluorescence of ZGO:Mn NRs indicated that the generation of ·O2 - was the main ROS species responsible for the photobleaching of ZGO:Mn NRs.

[0055] However, through experiments and detections, it was found that the theoretically maximum ·O2 - concentration in water was much lower than the amount required to completely quench ZGO:Mn NRs through static interaction; this indicated that static quenching by only ·O2 - could not fully explain the observed photobleaching. To separate ·O2 -For the contribution, the photo-bleached ZGO:Mn NRs were centrifuged and redispersed in fresh solution, and ·O2 was utilized - With a higher water solubility than O2, the fluorescence intensity only recovered 20 - 40% of the initial level after three cycles, indicating that other factors induced photo-bleaching beyond the quenching mediated by ·O2 - Further verification was carried out by time-resolved fluorescence decay monitoring. During a 30-minute dark interval, the fluorescence intensity only recovered 30% of the initial level, which can be explained by the dissipation of ·O2 - because of its extremely short lifetime in water. However, even after a long interruption, complete recovery could not be achieved, which confirmed that ·O2 - accounted for no more than 30% of the total photo-bleaching reason. This further jointly indicated that ·O2 - was not the main factor causing the photo-bleaching of ZGO:Mn NRs.

[0056] In-depth exploration of the experimental results in Example 3:

[0057] After more in-depth research and analysis, the following tentative photo-bleaching mechanism was proposed: After irradiation, ZGO:Mn NRs absorb high-energy photons with energy exceeding the bandgap, generating electron-hole pairs (conduction band electrons, e - ; valence band holes, h + ). These electron-hole pairs are initially trapped by the intrinsic crystal defects, forming metastable trap states. Subsequently, the slow release and recombination of the carriers trapped at the Mn 2+ luminescence centers generate long-lasting luminescence. However, the conduction band electrons can also reduce the surface-adsorbed O2 to ·O2 - , which statically quenches ~30% of the emission through the formation of non-luminescent complexes. At the same time, electron depletion leads to the accumulation of holes (h + ) within the NR lattice. This charge imbalance disrupts the radiative transition and inhibits photoluminescence. Importantly, the intrinsic defects of the NRs stabilize the trapped holes, exacerbate non-radiative recombination, and drive the remaining ~70% of the photo-bleaching. The accumulated photo-generated holes also explain why ZGO:Mn NRs are more susceptible to photo-bleaching than traditional luminescent materials despite their excellent long-afterglow performance.

[0058] Figure 7 a shows the reaction kinetic curve after adding 12 μM HGB to ZGO:Mn NRs prepared under the condition of pH 9.5 in Example 1, diluted to 0.12 mg / ml and photo-bleached; the dynamic response measurement confirmed the rapid stabilization of the signal within 3 minutes. Mechanism studies showed that HGB restored luminescence by neutralizing the photo-bleaching driving factors;

[0059] Figure 7b is the ZGO:Mn NRs prepared under the condition of pH 9.5 in Example 1, diluted to 0.12 mg / ml and photobleached. The Zeta potential of ZGO:Mn NRs after adding 12 μM HGB; The figure shows a significant change from -1.50 eV (after photobleaching) to -4.27 eV (HGB treatment), and it recovers near the potential before irradiation (-4.33 eV), indicating that the inhibition of the non-radiative pathway mediated by HGB may be to scavenge photogenerated holes (h+) and ROS;

[0060] Figure 7 c is the EPR spectrum of the DMPO-·O2 - signal after photobleaching and adding HGB; Figure 7 d is the ERP signal spectrum of TEPMO-h + The EPR spectrum confirms that after adding HGB, the ·O2 - ( Figure 7 c) and h + ( Figure 7 d) signals are eliminated, which means that a series of reactions occur among HGB, ·O2 - and photogenerated holes, and electron transfer may be involved.

[0061] A reasonable mechanism for the hemoglobin-enhanced luminescence of photobleached ZGO:Mn NRs is described as follows:

[0062]

[0063] HGB-Fe 2+ +O2→HGB-Fe 2+ …O2 (3)

[0064] ZGO:Mn * →ZGO:Mn+hv2 (4)

[0065] Analysis is as follows: Hemoglobin first coordinates with Zn 2+ ions on the surface of photobleached ZGO:Mn NRs through hydroxyl groups, promoting its adsorption (Equation 1). The strong oxidation potential of photogenerated holes (h + ) oxidizes the ferrous ion (Fe 2+ -HGB) of hemoglobin to ferric ion (Fe 3+ -HGB), neutralizes h + and restores the radiative recombination in ZGO:Mn NRs (Equation 4). Then, the Fe 3+ -HGB complex reacts with ·O2 - , accepts electrons to regenerate Fe 2+ -HGB, and at the same time decomposes ·O2 - into molecular oxygen (O2), thus eliminating ·O2 -mediated electrostatic quenching (Equation 2). Finally, the regenerated O2 binds to Fe 2+ -HGB to form a stable HGB-Fe 2+ -O2 adduct, thus preventing further photobleaching (Equation 3). Under this possibility analysis, the experimental results of Examples 2 and 3 can be understood.

[0066] Example 4

[0067] Continue to use the ZGO:Mn NRs prepared under the conditions of pH 9.5 in Example 1, dilute them to 0.12 mg / ml and divide them into two groups (fluorescent group, persistent luminescence group), 12 samples each, and irradiate them with 254 nm ultraviolet light for 600 seconds for photobleaching. Among the samples in the two groups, except for the blank samples, the other 11 samples were respectively added with L-cysteine, L-leucine, glutamic acid, L-proline, L-phenylalanine, glycine, L-norvaline, L-histidine, glutathione, bovine serum albumin, and serum to 6 μM; use a fluorescence spectrometer (Hitachi F-7100) to synchronously collect the fluorescence and persistent luminescence intensity data of ZGO:Mn NRs, and the parameter adjustment is the same as in Example 1. As Figure 8 shown in a, the left side of each pair of column charts in each group is the test result. It can be seen that these common blood components (amino acids, proteins) do not cause the luminescence reaction of photobleached ZGO:Mn NRs and have good selectivity for hemoglobin;

[0068] Next, add HGB to 6 μM in the 12 samples of the two groups respectively, and use the fluorescence spectrometer to collect the fluorescence and persistent luminescence intensity data again, with the parameter adjustment unchanged. As Figure 8 shown in a, the right side of each pair of column charts in each group is the test result. The fluorescence and long persistent luminescence ratios (I 536 / I 536,0 ) increased by 6.0 times and 11.0 times respectively, and the responses of other analytes were negligible. Visual inspection under ultraviolet excitation confirmed this specificity. Only hemoglobin triggered a bright green emission, while amino acids / proteins did not cause color changes. It should be noted that the hemoglobin detection is not affected by coexisting biomolecules, demonstrating that photobleached ZGO:Mn NRs have excellent selectivity and anti-interference ability in detecting HGB.

[0069] Example 5

[0070] Ultra-sensitive hemoglobin detection in aqueous environment: Prepare a 1% sodium alginate solution, and then add the ZGO:Mn NRs prepared under the condition of pH 9.5 in Example 1 to ensure that the concentration of ZGO:Mn NRs is 0.12 mg / mL. Mix well using a magnetic stirrer. Prepare a 0.18 mol / L calcium chloride solution. Use a dropper to suck an appropriate amount of the ZGO:Mn NRs and sodium alginate mixed solution and drop it into the calcium chloride solution to obtain fluorescent hydrogel beads. Irradiate the hydrogel beads with a 254 nm ultraviolet lamp for 10 min until their fluorescence quenches, and then place the hydrogel beads in an HGB solution with a concentration of 0 - 10 μM and let it stand, and then take out and take a fluorescence photo. As Figure 9 shown, when immersed in the hemoglobin solution, the green fluorescence of the photo-bleached ZGO:Mn NRs recovers. Therefore, this property can be used to detect trace amounts of hemoglobin in water.

[0071] Use the ZGO:Mn NRs prepared under the condition of pH 9.5 in Example 1, dilute it with water to 0.12 mg / mL, and irradiate it with a 254 nm ultraviolet lamp for 10 min until its fluorescence quenches (hereinafter referred to as photo-bleached ZGO:Mn NRs in the following experiments and Example 6). Add real blood, and measure the fluorescence intensity at different stages during the experiment; as Figure 10 shown, it can be seen from the change of the fluorescence intensity of the photo-bleached ZGO:Mn NRs solution after exposure to real blood that the fluorescence of the photo-bleached ZGO:Mn NRs increases significantly after adding real blood.

[0072] In addition, since hemoglobin is a key component of blood, this method can also be used to detect potential blood stains that are invisible in sunlight. Refer to Figure 11 , use a footprint stamp to dip into diluted real blood (0%, 0.01%, 0.02%, 0.1%, 0.2%, 1%) and stamp it on the surface to be detected. After treatment with photo-bleached ZGO:Mn NRs, blood diluted to as low as 0.01% can be clearly identified, thus revealing potential blood stains; therefore, as long as there is residual hemoglobin, whether it is fresh or old blood stains, photo-bleached ZGO:Mn NRs can be identified.

[0073] As Figure 12As shown, a footprint stamp was used to dip an appropriate amount of bloodstains and print them on the surfaces of different substrates respectively to form clear bloodstain imprints, including non-porous materials and porous substrates. The markings in the figure are explained as follows: floor - ceramic tile, desktop - wooden board, foamboard - foam board, cotton fiber - cotton fiber, wall - concrete block with white wall paint on the surface, plasticboard - plastic board, garbage bag - plastic bag. Immediately afterwards, the substrate samples with printed bloodstains were placed in an oven for drying. The oven temperature was set at 100 °C and the drying time was 1 h to ensure that the dried bloodstains showed no obvious signs of wetness and the imprints were stable. The photo - bleached ZGO:Mn NRs were diluted with deionized water and filled into a spray bottle, and evenly sprayed on the surface of the dried substrates to ensure complete coverage of the bloodstain pattern. Then the treated substrates were placed under a 254 nm ultraviolet lamp for irradiation, and image acquisition was carried out using a Canon camera. The results confirmed that the photo - bleached ZGO:Mn NRs could be normally applied to HGB detection on the surfaces of various materials. Even in the presence of autofluorescence interference, the shape of the bloodstains on the shoe prints was still clearly distinguishable from the background.

[0074] Example 6

[0075] A key challenge in bloodstain detection is the false positives of hypochlorous acid and sodium hypochlorite, which are also common disinfectant components. In this example, the method corresponding to dipping different concentration samples with a footprint stamp in Example 5 was adopted, and detection and comparison were carried out using photo - bleached ZGO:Mn NRs. The samples were 6 μM HGB aqueous solution, 3 μM hypochlorous acid aqueous solution, 6 μM hypochlorous acid aqueous solution, 20 μM hypochlorous acid aqueous solution, 60 μM hypochlorous acid aqueous solution, and a commercially available 84 disinfectant (5% sodium hypochlorite solution diluted 1:100) after simulated dilution; Figure 11 It was found through testing that although all hypochlorous acid and sodium hypochlorite samples initially simulated the fluorescence recovery induced by hemoglobin, these samples quickly showed attenuation. Refer to

[0076] Figure, where the upper part of the figure is the graph of the fluorescence signal of 6 μM HGB changing with time, and the lower part is the graph of the fluorescence signal of 6 μM hypochlorous acid aqueous solution changing with time. Its signal decayed to the baseline within 110 seconds, in sharp contrast to the stable luminescence of the samples containing HGB. All the above hypochlorous acid and sodium hypochlorite samples decayed to the point where they were difficult to distinguish with the naked eye within 600 seconds. Similarly, the long - afterglow persistent luminescence characteristics of all samples were also restored at the beginning, but the long - afterglow persistent luminescence of hypochlorous acid and sodium hypochlorite samples decayed rapidly after continuous irradiation with the excitation light. Figure 13

[0077] Utilizing the characteristics of this example, through integrated time - gated detection, the transient interference of hypochlorous acid and sodium hypochlorite can be effectively suppressed, making the destruction of common bleaching agents at the crime scene a thing of the past and improving the reliability of bloodstain detection.​

[0078] In order to verify this application, the Figure 12 Two groups of four dry samples (ceramic tiles, wooden boards, aluminum alloy plates, and plastics) were prepared using the corresponding method. The footprints of the first group were dipped in real blood, and the footprints of the second group were dipped in 12 μM hypochlorous acid solution. After drying at 100 ° C for 1 hour and cooling to room temperature, commercially available Diao Pai 84 disinfectant was diluted with tap water at a ratio of 1:100 and then sprayed on the surface of the eight dried samples. The footprints were scrubbed with a hard brush until they were just invisible to the naked eye. The sample surface was blotted dry with absorbent paper (note that it cannot be wiped back and forth, only static blotting is allowed). Then, the ZGO:Mn NRs were sprayed to cover 8 samples. Under darkroom conditions, under 254nm ultraviolet irradiation, the entire surface of the 8 samples emitted green fluorescence, and footprints could not be distinguished. After 10 seconds of 254nm ultraviolet irradiation, it was turned off and the photon camera was used to observe that the entire surface of the 8 samples continued to glow, and footprints could not be distinguished. The 8 samples were continuously irradiated with 254nm ultraviolet. After about 200 seconds, the 4 samples in the first group were fluorescent on the entire surface, but footprints were vaguely visible to the naked eye. The fluorescence level of the second group was relatively even, and footprints could not be distinguished. After turning off the ultraviolet, the photon camera was used to observe that all 8 samples continued to glow and it was difficult to distinguish footprints. The 254nm ultraviolet irradiation was continued for 8 samples. The samples were observed after a total of 600 seconds of irradiation. The entire surface of the 8 samples still had fluorescence, but only the 4 samples in the first group showed easily identifiable footprints. The UV was turned off and the photon camera was used to image the 8 samples, and it was observed that all had long afterglow and continued to glow, but the footprints of the 4 samples in the first group were obviously brighter than the surrounding areas; the 8 samples were continuously irradiated with 254nm UV light, and the samples were observed after a total of 30 minutes of irradiation. The fluorescence of the second group almost disappeared, and the footprints of the 4 samples in the first group had significant fluorescence, but there was still visible fluorescence in the surrounding areas without footprints (which should be related to the spread of blood residue during brushing). The UV was turned off and the photon camera was used to image the 8 samples, and it was observed that only the footprints of the 4 samples in the first group continued to glow.

[0079] Experiments show that both hypochlorous acid and sodium hypochlorite can restore the fluorescence and persistent luminescence properties of photobleached ZGO:Mn NRs, which are both induced by hypochlorite. However, the rate at which the fluorescence and persistent luminescence decay over time is significantly faster than the rate at which the hemoglobin-induced fluorescence and persistent luminescence decay after recovery. This also confirms the mechanism of hemoglobin-enhanced luminescence of photobleached ZGO:Mn NRs explored after Example 3. This property can be used to identify and distinguish residual blood stains that have been interfered with by bleaching agents containing hypochlorous acid or sodium hypochlorite.

[0080] In addition to the above-mentioned studies on the fluorescence characteristics of photobleached ZGO:Mn NRs after reaction with hemoglobin, during the experiment of Example 1, it was observed that the trend of the sustained luminescence decay curve was the same as that of the fluorescence decay curve. Example 2 also gave the linear range and detection limit of the sustained luminescence ( Figure 4 c andFigure 4 d), Example 3 also records the long-term duration of persistent luminescence ( Figure 5 b), The two groups of experiments in Example 4 also include the selectivity and anti-interference tests of persistent luminescence ( Figure 8 a), In fact, the application of the manganese-doped zinc germanate nanorod fluorescent probe of the present invention in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis is not limited to the fluorescence recovery after the reaction of photo-bleached ZGO:Mn NRs with hemoglobin. The persistent afterglow luminescence characteristics of itself after the reaction also recover. According to Figure 5 b, After the reaction, in addition to observing fluorescence under ultraviolet irradiation, persistent afterglow luminescence can also be observed by means of photon camera imaging, etc. Even the imaging parameters of the same device and the brightness of the images after imaging can be established into a result comparison experience table within a roughly range, so as to make a preliminary judgment on the hemoglobin concentration; The persistent luminescence recorded in the present invention is persistent afterglow or phosphorescence.

[0081] In addition, although the above examples only record the experimental data of photo-bleached ZGO:Mn NRs prepared under the condition of pH 9.5, it can be known from Example 1 that at other pH values, only the attenuation amounts of fluorescence and persistent luminescence after photo-bleaching are less than those at pH 9.5. Similarly, changes in the parameters of the preparation method, or whether alkalization is carried out, also affect the photo-bleaching attenuation, but will not change the intrinsic characteristics of ZGO:Mn NRs; Although the above examples mostly use 254 nm ultraviolet light to irradiate for 10 min for photo-bleaching, ultraviolet light with wavelengths of 100 - 365 nm is effective for photo-bleaching, and the irradiation duration can be 1 - 30 min, among which 200 - 312 nm is better, but only the photo-bleaching efficiency and other aspects are different, and it is not necessary to fix this condition. Since the conclusion explored in the present invention is a series of reactions occurring between HGB, ·O2 - and photogenerated holes, the conditions that can achieve photo-induced fluorescence quenching and generate photogenerated holes can be applied to the present invention, and it is not limited to ultraviolet irradiation. That is, in the future, new conditions for photo-induced fluorescence quenching and generating photogenerated holes may also appear, and it should also be understood that they are within the protection scope of the photo-bleached ZGO:Mn NRs recorded in the present invention; For the detection limit and linear range in Example 2, they are not the key content expressed in the present invention. This example only illustrates that the photo-bleached ZGO:Mn NRs can identify trace hemoglobin; The 20 minutes and 2 days in Example 3 are not the maximum durations of fluorescence and persistent luminescence after the reaction continues to be exposed to light and environmental oxygen. The actual observable effective time is much longer than 20 minutes and 2 days. From Figure 5The slowdown of the downward trend can also be used for corresponding theoretical judgments; although only 11 substances are listed in the selectivity and anti-interference experiments in Example 4, this is not a limitation on the photo-bleached ZGO:Mn NRs. It is just an example, and the actual selective and anti-interference substances cannot be exhausted; the several experiments in Example 5 are also illustrations of the possibilities of the application scope, rather than absolute limitations; the concentration and material in Example 6 are also for illustration, only to show that under the condition of mixing HGB with hypochlorous acid and sodium hypochlorite, the photo-bleached ZGO:Mn NRs can identify and distinguish, and it should be understood that other substances containing hypochlorite ions, if the principle is the same as that of hypochlorous acid and sodium hypochlorite, also conform to the use of the fluorescence and the decay rate after persistent luminescence recovery proposed by the present invention to distinguish from hemoglobin; the parameters detected by the fluorometer in each example should not be limited to the records in Example 1, and the emission wavelength of 300-700 nm is acceptable, and the adjustment of the observed parameters is not the focus of the present invention either.

[0082] Therefore, it should be understood that the above-mentioned examples are mainly for illustration rather than limitation. The above are only the preferred embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope defined by the claims.

[0083] Where the present invention is not described in detail, it is the well-known technology of those skilled in the art.

Claims

1. Application of manganese-doped zinc germanate nanorod fluorescence probe in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis, characterized in that: The photo-bleached zinc germanate nanorod fluorescent probe doped with manganese restores its fluorescence and persistent luminescence properties after contacting a sample containing hemoglobin, and hemoglobin can prevent the re-photo-bleaching of the zinc germanate nanorod fluorescent probe doped with manganese; the photo-bleached zinc germanate nanorod fluorescent probe doped with manganese restores its fluorescence and persistent luminescence properties after contacting a sample containing hypochlorous acid or sodium hypochlorite, but the decay rate of the fluorescence and persistent luminescence over time is greater than the decay rate after the fluorescence and persistent luminescence are restored induced by hemoglobin.

2. Application of the manganese-doped zinc germanate nanorod fluorescence probe according to claim 1 in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis, characterized in that: The sample containing hemoglobin includes a sample mixed with hypochlorous acid or sodium hypochlorite.

3. Application of the manganese-doped zinc germanate nanorod fluorescence probe according to claim 1 in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis, characterized in that: The sample containing hemoglobin includes undiluted real blood and diluted blood.

4. Use of the manganese-doped zinc germanate nanorod fluorescence probe according to claim 1 in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis, characterized in that: The sample containing hemoglobin includes fresh or old latent bloodstains invisible under sunlight.

5. Use of the manganese-doped zinc germanate nanorod fluorescent probe according to claim 1 in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis, characterized in that: The sample containing hemoglobin is from different material surfaces.

6. Use of the manganese-doped zinc germanate nanorod fluorescence probe according to claim 1 in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis, characterized in that: The photo-bleaching is ultraviolet irradiation at 100 - 365 nm for 1 - 30 min.

7. Use of the manganese-doped zinc germanate nanorod fluorescence probe according to claim 1 in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis, characterized in that: The preparation method of the zinc germanate nanorod fluorescent probe doped with manganese includes mixing Zn(NO3)2, Mn(NO3)2 and concentrated nitric acid, adding them to deionized water and stirring vigorously, then adding a Na2GeO3 solution, quickly adding ammonium hydroxide to adjust the pH of the system to 6 - 10 under vigorous stirring, then transferring the reaction solution to a reaction kettle and continuing to stir at room temperature for 1 - 24 h, and then placing the reaction kettle at 100 - 300 °C for reaction for 4 - 24 h, and centrifugally washing the product with deionized water to obtain ZGO:Mn NRs.

8. Application of manganese-doped zinc germanate nanorod fluorescence probe in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis, characterized in that: After the zinc germanate nanorod fluorescent probe doped with manganese is photo-bleached, it is used for detecting the residual bloodstains invisible to the naked eye under sunlight after being cleaned with hypochlorous acid or sodium hypochlorite at the crime scene.

9. Use of the manganese-doped zinc germanate nanorod fluorescence probe according to claim 8 in anti-hypochlorous acid and sodium hypochlorite interference in blood analysis, characterized in that: The detection method is to cover the area to be detected with the solution of the photo-bleached zinc germanate nanorod fluorescent probe doped with manganese, continuously irradiate with the excitation light, observe whether the fluorescence intensity is higher than that of the surrounding area after the fluorescence decays over time, or turn off the excitation light after irradiating with the excitation light, and collect and observe whether the persistent luminescence intensity is higher than that of the surrounding area after the persistent afterglow decays.

Images