Carbon quantum dots preparation method and application thereof in detection of trivalent iron ion in water quality sample

Abstract

The application provides a carbon quantum dot preparation method and application thereof in trivalent iron ion detection of a water quality sample. The carbon quantum dots are prepared by using specific microbial strains through activation, freeze-drying and hydrothermal synthesis, wherein the polyhydroxybutyrate engineering bacteria can be doped with S-PHB-CQDs. The prepared carbon quantum dots are used for water quality Fe 3+ Detection: the detection limit of PHB-CQDs is 0.507 μmol / L (pH 3-11); the detection limit of S-PHB-CQDs is 0.997 μmol / L (pH 0-3), and the sensitivity is improved by 14 times compared with the wild type. The carbon quantum dots are integrated into a paper-based device to realize portable semi-quantitative detection. The carbon quantum dots have the advantages of environmental protection, high sensitivity and wide pH adaptability.

Description

TECHNICAL FIELD

[0001] The present application relates to the cross field of biological nanomaterial synthesis technology and environmental analysis and detection technology, and particularly relates to a carbon quantum dot preparation method and application thereof in trivalent iron ion detection of water quality samples. BACKGROUND

[0002] Carbon quantum dots (CQDs), as zero-dimensional carbon-based nanomaterials (1-10 nm), have attracted extensive attention since their discovery in 2004 due to their unique photoluminescence properties, high water solubility, tunable surface chemistry, and excellent biocompatibility. Unlike traditional heavy metal quantum dots (such as CdSe, PbS) with toxicity, CQDs have significant advantages such as low toxicity, environmental sustainability, and cost-effectiveness.

[0003] The presence of surface carboxyl and hydroxyl plasma functional groups in CQDs enables them to generate high osmotic pressure, enhancing water flux while minimizing solute reverse osmosis. These characteristics, combined with their low toxicity and biocompatibility, make CQDs an environmentally friendly alternative to traditional chemical solutes. In addition, CQDs have been widely explored in multiple fields such as bioimaging, optoelectronic devices, environmental sensing, and catalytic systems. Notably, their excitation-dependent fluorescence emission properties and surface functionalization potential make CQDs ideal candidate materials for developing next-generation fluorescent probes. Metal ion detection sensors based on CQDs, especially Fe 3+ detection sensors have become promising alternatives to traditional methods, which often require expensive equipment and complex pretreatment procedures. However, the green and efficient synthesis of CQDs for high-sensitivity and selective Fe 3+ detection still faces significant challenges.

[0004] Currently, CQDs are mainly synthesized from biomass, plant waste or lignin and other low-cost, high-storage renewable resources in a green economic way. However, these synthesis processes are usually tedious and have low yield, which significantly limits the practical application of CQDs. In recent years, microorganisms have become excellent precursors for the synthesis of CQDs. The existing technology reports the development of nanoscale fluorescent materials with excellent performance using microbial biomass. For example, blue-green algae-derived CQDs exhibit excellent photo-stability in metal ion sensing and have been integrated into composite films with hydrophobicity and enhanced ultraviolet (UV) and infrared radiation shielding capability. In addition, a variety of microorganisms have been explored for multifunctional applications: Arthrospira platensis shows potential for efficient degradation of textile wastewater, marine endophytic fungi (Aspergillus flavus) can increase curcumin production, and Lactobacillus casei biomass for producing lactic acid has been used to synthesize CQDs with excellent water solubility, photophysical properties and tetracycline detection potential. Despite these advances, the synthesis of microorganism-derived CQDs still faces the challenges of complex procedures and low yield, which greatly limits their widespread application. Therefore, further research and development of microorganism-derived CQDs with high fluorescent performance and higher synthesis efficiency are needed considering the abundance of resources, ideal performance and economic feasibility.

[0005] Zymomonas mobilis, as a natural ethanol-producing bacterium, has become an excellent chassis microorganism for biochemical production due to its excellent sugar conversion efficiency and environmental adaptability. Notably, DuPont has used Zymomonas mobilis as a microbial platform to establish a commercial-scale cellulosic bioethanol plant with an annual production capacity of 83,000 tons. With the development of systems biology and synthetic biology, an improved iZM516 genome-scale metabolic model, multifunctional biological elements, high-efficiency genome editing tools and related repair pathways have been constructed and developed. Therefore, a series of microbial cell factories have been constructed for the production of various platform compounds in Zymomonas mobilis, including D-lactic acid, poly-3-hydroxybutyrate (PHB, same below), L-serine, acetoin, 2,3-butanediol (2,3-BDO), isobutanol, farnesene and ethylene, etc. All these products produced by engineered Zymomonas mobilis provide a variety of resources for the synthesis of microbial-derived CQDs (Mic-CQDs) due to the presence of different ionic functional groups in the rich products (such as proteins, lipids, hopanoids and exopolysaccharides, etc.), especially the functional groups accumulated in the cells. However, the existing technology rarely uses engineered microorganisms to synthesize CQDs, and even less has been applied to Fe 3+ detection. SUMMARY

[0006] The microbial strains used in the present application are all wild-type ZM4, ZMNP or polyhydroxybutyrate-producing engineering strain ZMPt-Flo of Zymomonas mobilis producing ethanol N2-EUPOne of the above, in the classification of bacteria, all belong to different strains of Zymomonas mobilis. Among them, the wild type ZM4 of ethanol-producing Zymomonas mobilis is the standard model strain Z.mobilis ZM4 ATCC 31821 (Seo J S, Chong H, Park H S, et al. The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4), which can be purchased through commercial channels; ethanol-producing Zymomonas mobilis ZMNP (Geng B, Wu Y, Wu X, et al. Efficient genome-editing tools to engineer the recalcitrant non-model industrial microorganism Zymomonas mobilis) and polyhydroxybutyrate-producing engineering bacteria ZMPt-Flo N2-EUP (Li Y, Wang Y, Wang R, et al. Metabolic engineering of Zymomonas mobilis for continuous co-production of bioethanol and poly-3-hydroxybutyrate (PHB)) The original source is obtained by experimental technical means by the research group, and the public can obtain relevant germplasm resource materials from the applicant, which can only be used for repeated experimental use of the application and cannot be used for other purposes.

[0007] The purpose of the present application is to provide a carbon quantum dot preparation method, which mediates the formation of carbon quantum dots by intracellular biological materials of engineering strains, realizes precise surface engineering of carbon quantum dots (CQDs), and integrates into colorimetric paper chips, which can be applied to on-site semi-quantitative detection of ferric ion in water samples.

[0008] In the first aspect, the present application provides a carbon quantum dot preparation method, comprising the following steps:

[0009] SA-1, activating and expanding the microbial strain to obtain a bacterial liquid;

[0010] SA-2, freezing and drying the bacterial liquid in step SA-1 after centrifugation and washing to obtain a bacterial dry powder;

[0011] SA-3, synthesizing carbon quantum dots from the bacterial dry powder in step SA-2 by hydrothermal synthesis method.

[0012] Preferably, the microbial strain is one of ZM4, ZMNP, ZMPt-Flo N2-EUP .

[0013] Further preferably, the microbial strain is ZMPt-Flo N2-EUP .

[0014] Preferably, the hydrothermal synthesis method comprises the following steps: 1g of bacterial dry powder is homogeneously mixed with deionized water at a mass-volume ratio of 1g:30mL, and then placed in a reaction kettle, and hydrothermally reacted at 200-220℃ for 6-8h.

[0015] Or, 1g of bacterial dry powder is reacted with 30mL of sulfuric acid with a mass concentration of 60% at 90-100℃ for 20-30min, and then placed in a reaction kettle, and twice carbonized at 200-220℃ for 6-8h.

[0016] In a second aspect, the present application also provides a carbon quantum dot prepared by the above preparation method.

[0017] In a third aspect, the present application also provides an application of the above carbon quantum dot in the detection of Fe3+ in a water sample, comprising the following steps:

[0018] SB-1, pre-mixing the sample to be tested with the above carbon quantum dot solution;

[0019] SB-2, measuring the fluorescence intensity F0 of the carbon quantum dot solution without Fe 3+ at the maximum ultraviolet excitation wavelength;

[0020] SB-3, measuring the fluorescence intensity F of the sample pre-mixing solution of step SB-1 at the maximum ultraviolet excitation wavelength;

[0021] SB-4, calculating the Fe 3+ concentration according to the calibration curve equation of the normalized quenching ratio (F0-F) / F0 and the Fe 3+ concentration (μmol / L).

[0022] Preferably, when the carbon quantum dot is PHB-CQDs, the pH range of the sample to be tested is 3-11.

[0023] Preferably, when the carbon quantum dot is S-PHB-CQDs, the pH range of the sample to be tested is 0-3.

[0024] In a fourth aspect, the present application also provides a Fe 3+ detection paper chip, which is composed of a paper-based carrier and the above carbon quantum dot.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] (1) S-PHB-CQDs achieve the highest fluorescence quantum yield (40.1%) of precursor carbon quantum dots with microorganisms as carbon sources.

[0027] (2) PHB-CQDs as Fe 3+ fluorescent sensors, Fe 3+ linear range (2.5-100 μmol / L) is sensitive and extremely consistent with the water quality detection range, and S-PHB-CQDs as Fe 3+ fluorescent sensors solve the problem that conventional carbon quantum dots cannot be detected in a strong acidic environment.

[0028] (3) The surface functional groups of carbon quantum dots are modified for the first time by using intracellular materials of microorganisms. Compared with carbon quantum dots (hereinafter referred to as ZM4-CQDs) synthesized by wild-type strain ZM4 (linear range 20-75 μmol / L, detection limit 7.16 μmol / L, R 2 = 0.994), the detection sensitivity of PHB-CQDs (linear range 2.5-100 μmol / L, detection limit 0.507 μmol / L, R 2 = 0.998) to Fe 3+ is increased by 14 times.

[0029] (4) Microbial carbon quantum dots PHB-CQDs are integrated into a paper-based detection device, which is convenient for on-site semi-quantitative detection. The optimized carbon quantum dots exhibit excellent recovery rate in actual water samples, and are successfully integrated into a colorimetric paper chip for on-site Fe 3+ monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0031] Figure 1 It is a schematic diagram of the one-step hydrothermal synthesis preparation method of microbial derived CQDs in Example 1.

[0032] Figure 2 It is a transmission electron microscope (TEM) characterization diagram of the morphology of the three strains of microorganisms in Performance Characterization and Performance Test 1. Among them, (a) is the strain ZM4, (b) is the strain ZMNP, and (c) is the strain ZMPt-Flo N2-EUP .

[0033] Figure 3 Fig. 1 is a morphology characterization diagram of the five kinds of microbial-derived CQDs in performance characterization and performance test 1.; wherein, (a) is the ZM4-CQDs, (b) is the ZMNP-CQDs, (c) is the PHB-CQDs, (d) is the PHB dopant -CQDs, and (e) is the S-PHB-CQDs.

[0034] Figure 4 Fig. 2 is a chemical structure characterization diagram of the five kinds of microbial-derived CQDs in performance characterization and performance test 2.; wherein, (a) is the FT-IR spectrum, and (b) is the XPS spectrum full scan spectrum.

[0035] Figure 5 Fig. 3 is an XPS spectrum high-resolution C1s peak spectrum diagram of the five kinds of microbial-derived CQDs in performance characterization and performance test 2.; wherein, (a) is the ZM4-CQDs, (b) is the ZMNP-CQDs, (c) is the PHB-CQDs, (d) is the PHB dopant -CQDs, and (e) is the S-PHB-CQDs.

[0036] Figure 6 Fig. 4 is an XPS spectrum high-resolution O1s peak spectrum diagram of the five kinds of microbial-derived CQDs in performance characterization and performance test 2.; wherein, (a) is the ZM4-CQDs, (b) is the ZMNP-CQDs, (c) is the PHB-CQDs, (d) is the PHB dopant -CQDs, and (e) is the S-PHB-CQDs.

[0037] Figure 7 Fig. 5 is an XPS spectrum high-resolution N1s peak spectrum diagram of the four kinds of microbial-derived CQDs in performance characterization and performance test 2., and an XPS spectrum high-resolution S2p peak spectrum diagram of the S-PHB-CQDs; wherein, (a)-(d) are respectively the XPS spectrum high-resolution N1s peak spectrum diagram of the ZM4-CQDs, the ZMNP-CQDs, the PHB-CQDs, and the PHB dopant -CQDs, and (e) is the XPS spectrum high-resolution S2p peak spectrum diagram of the S-PHB-CQDs.

[0038] Figure 8 Fig. 6 is a SEM image of the hydrothermal synthesis residues of the five kinds of microbial-derived CQDs in performance characterization and performance test 2.; wherein, (a) is the ZM4-CQDs, (b) is the ZMNP-CQDs, (c) is the PHB-CQDs, (d) is the PHB dopant -CQDs, and (e) is the S-PHB-CQDs.

[0039] Figure 9 Predicted structure schematic of ZM4-CQDs, ZMNP-CQDs and PHB-CQDs for performance characterization and performance test 2.

[0040] Figure 10 UV-Vis spectra and down-conversion fluorescence spectra of 5 kinds of microbial derived CQDs for performance characterization and performance test 3; wherein, (a) figure is ZM4-CQDs, (b) figure is ZMNP-CQDs, (c) figure is PHB-CQDs, (d) figure is PHB dopant -CQDs, (e) figure is S-PHB-CQDs.

[0041] Figure 11 Relative fluorescence quantum yield (QY) calculation of 5 kinds of microbial derived CQDs for performance characterization and performance test 3.

[0042] Figure 12 Fluorescence lifetime spectrum of 5 kinds of microbial derived CQDs for performance characterization and performance test 3.

[0043] Figure 13 Schematic diagram of static fluorescence quenching mechanism between PHB-CQDs and Fe 3+ for performance characterization and performance test 4.

[0044] Figure 14 Influence of 13 kinds of metal ions on PHB-CQDs as Fe 3+ blue fluorescence detection probe under 350 nm excitation for performance characterization and performance test 4; wherein, (a) figure is the normalized fluorescence intensity of PHB-CQDs solution after adding 5 mmol / L concentration of 13 kinds of metal ions, (b) figure is the fluorescence response of PHB-CQDs solution when 0.5 mmol / L Fe 3+ and other metal ions are added at the same time (M n+ represents each metal ion), (c) figure is the fluorescence quenching of CQDs solution with the increase of Fe 3+ concentration (0-5 mmol / L).

[0045] Figure 15 Fluorescence quenching degree of 5 kinds of microbial derived CQDs as Fe 3+ blue fluorescence detection probe and Fe 3+ concentration for performance characterization and performance test 4; wherein, the inset shows their linear detection range of Fe 3+ .

[0046] Figure 16The results of the pH stability test of the five kinds of microbial-derived CQDs in Performance Characterization and Performance Test 4.

[0047] Figure 17 The results of the pH stability test of the five kinds of microbial-derived CQDs in Performance Characterization and Performance Test 4. 3+ Detecting the paper chip. When detecting the sample, the fluorescence color of the paper chip gradually changes from blue to black. DETAILED DESCRIPTION

[0048] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0049] It should be noted that the order of the following embodiments is not limited as the preferred order of the embodiments. In addition, in the description of the present application, the term "comprising" means "including but not limited to". Various embodiments of the present application can exist in the form of a range; it should be understood that the description in the form of a range is only for the convenience and brevity, and should not be understood as a hard limitation on the scope of the present application; therefore, it should be considered that the described range has been specifically disclosed all possible sub-ranges and single values within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, regardless of the range. In addition, whenever a numerical range is indicated in this document, it means that any cited number (fraction or integer) within the indicated range is included.

[0050] The present application provides a carbon quantum dot preparation method, comprising the following steps:

[0051] SA-1, activating and expanding the microbial strain to obtain a bacterial liquid;

[0052] SA-2, freezing and drying the bacterial liquid in step SA-1 after centrifugation and washing to obtain a bacterial dry powder;

[0053] SA-3, synthesizing carbon quantum dots from the bacterial dry powder in step SA-2 by a hydrothermal synthesis method.

[0054] Specifically, the microbial strain is one of ZM4, ZMNP and ZMPt-Flo. N2-EUP

[0055] ​Further specifically, the microbial strain is polyhydroxybutyrate-producing engineered bacteria ZMPt-Flo N2-EUP .

[0056] Further specifically, the microbial strain is cultured in RMG5 medium to obtain a bacterial solution.

[0057] More specifically, the RMG5 medium has the following formulation: 50 g / L glucose, 10 g / L yeast extract (Thermo Fisher Scientific Inc., Catalog No. LP0021B), 2 g / L KH2PO4.

[0058] Specifically, the hydrothermal synthesis method comprises the following steps: 1 g of bacterial dry powder is homogeneously mixed with deionized water at a mass-volume ratio of 1 g:30 mL, and then placed in a reaction kettle, and hydrothermal reaction is carried out at 200-220℃ for 6-8 h; or, 1 g of bacterial dry powder is reacted with 30 mL of sulfuric acid with a mass concentration of 60% at 90-100℃ for 20-30 minutes, and then placed in a reaction kettle, and secondary carbonization is carried out at 200-220℃ for 6-8 h.

[0059] Further specifically, the carbon quantum dots prepared after hydrothermal synthesis are crude products, and microbial carbon quantum dot powder is obtained after purification.

[0060] More specifically, the purification process comprises centrifugation, filtration, dialysis, extraction and freeze-drying processes.

[0061] The present application also provides a carbon quantum dot prepared by the above preparation method.

[0062] The present application also provides an application of the above carbon quantum dot in the detection of trivalent iron ions in water samples, which comprises the following steps:

[0063] SB-1, pre-mixing the sample to be tested with the above carbon quantum dot solution;

[0064] SB-2, measuring the fluorescence intensity F0 of the carbon quantum dot solution without Fe 3+ at the maximum ultraviolet excitation wavelength;

[0065] SB-3, measuring the fluorescence intensity F of the sample pre-mixing solution of step SB-1 at the maximum ultraviolet excitation wavelength;

[0066] SB-4, calculating the Fe 3+ concentration according to the calibration curve equation of the normalized quenching ratio (F0-F) / F0 and the Fe 3+ concentration (μmol / L).

[0067] Specifically, when the carbon quantum dots are PHB-CQDs, the pH range of the sample to be tested is 3-11.

[0068] Specifically, when the carbon quantum dots are S-PHB-CQDs, the pH range of the sample to be detected is 0-3.

[0069] The application further provides a Fe 3+ The detection paper chip is composed of a paper-based carrier and the aforementioned carbon quantum dots.

[0070] Specifically, the Fe 3+ The preparation method of the detection paper chip is as follows:

[0071] (1) 0.4 g of sodium carboxymethyl cellulose is dissolved in 10 mL of deionized water at 100 DEG C, 3 mL of 1% polyvinyl alcohol and 2 mL of 10% citric acid solution are added, and the mixture is cooled to 40-45 DEG C to obtain a paper-based carrier.

[0072] (2) PHB-CQDs are added to the paper-based carrier at 40-45 DEG C to obtain a Fe 3+ detection paper chip.

[0073] Specifically, the paper-based detection device needs to be used under the excitation of 350 nm ultraviolet light.

[0074] The carbon quantum dot preparation method and the application thereof in the detection of ferric ions in water samples are further described in the following specific examples. This part further describes the content of the application in combination with specific examples, but should not be understood as a limitation of the application. If not specifically stated, the technical means adopted in the examples is the conventional means familiar to those skilled in the art. Unless specifically stated, the reagents, methods and equipment adopted in the application are the conventional reagents, methods and equipment in the art.

[0075] In the following examples, the double exponential decay model is fitted by a double exponential function R(t):

[0076]

[0077] Wherein, R(t) represents the fluorescence decay signal at time t, t represents the time variable, A1 and A2 represent the amplitudes of the surface and the core, respectively, and τ1 and τ2 represent the characteristic lifetimes of the surface and the core, respectively.

[0078] In the following examples, the average fluorescence lifetime τ avg is calculated according to the following formula:

[0079]

[0080] Wherein, A1 and A2 represent the amplitudes of the surface and the core, respectively, and τ1 and τ2 represent the characteristic lifetimes of the surface and the core, respectively.

[0081] In the following examples, the relative photoluminescence quantum yield φ (QY) is calculated based on the following formula, with quinine sulfate (Macklin Inc., Catalog No.: Q817155) fluorescence standard solution (0.1 mol / L H2SO4, quantum yield 54%) as the reference standard:

[0082]

[0083] wherein subscript "x" represents the microbial source CQDs, subscript "std" represents the quinine sulfate fluorescence standard solution, η represents the refractive index (water solution η = 1.33), A is the absorbance at the excitation wavelength, and I is the integrated fluorescence intensity of the fluorescence emission spectrum (represented by the integrated area of the PL emission spectrum).

[0084] In the following examples, the normalized fluorescence intensity is calculated according to the following formula:

[0085]

[0086] wherein F o represents the fluorescence intensity of CQDs (control group) without Fe 3+ , and F represents the fluorescence intensity of CQDs (experimental group) after adding Fe 3+ .

[0087] In the following examples, the calculation formula of the detection limit (LOD) is as follows:

[0088]

[0089] wherein N represents the standard deviation of noise, and S represents the sensitivity (i.e. the ratio of signal response value to Fe 3+ concentration).

[0090] In the following examples, the percentage of each substance solution represents the mass concentration of the solution, and the calculation formula is as follows:

[0091]

[0092] Example 1 Preparation of microbial carbon quantum dots ZM4-CQDs

[0093] This example provides a preparation method of microbial carbon quantum dots ZM4-CQDs (as shown in Figure 1 ), which specifically comprises the following steps:

[0094] (1) Activating and expanding the microbial strain bacterial solution

[0095] The wild type ZM4 of Zymomonas mobilis was activated and expanded using RMG5 medium, and was cultured at 30°C with a shaking speed of 200 rpm for 24 h to obtain the bacterial solution.

[0096] (2) Preparation of bacterial dry powder

[0097] The bacterial solution obtained in step (1) was collected by using a Sorvall WX 80+ ultracentrifuge (Thermo Fisher Scientific Inc., 10,000 rpm, 10 minutes) to collect microbial cells, washed with phosphate buffer solution (PBS, Thermo Fisher Scientific Inc., Cat. No. 20012027), and then freeze-dried at -50°C for 48 hours using a freeze dryer (Beijing Yaxing Instrument Technology Development Co., Ltd., Model: LGJ-10N) to obtain a bacterial dry powder.

[0098] (3) Preparation of microbial carbon quantum dot crude product (hydrothermal synthesis)

[0099] 1 g of bacterial dry powder was homogeneously mixed with deionized water at a mass-volume ratio of 1 g:30 mL, and subjected to hydrothermal treatment at 200°C under water for 6 hours in a 50 mL polytetrafluoroethylene-lined stainless steel high-pressure reaction kettle (Shanghai Huotong Instrument Co., Ltd., HT-FC model) to obtain a carbon quantum dot crude product.

[0100] (4) Purification of microbial carbon quantum dot powder

[0101] The crude product was cooled to room temperature and centrifuged to obtain a deep amber supernatant. The supernatant was sterile-filtered using a 0.22 μm water-phase filter membrane (Tianjin Tengen Experimental Equipment Co., Ltd.), then dialyzed against deionized water in a cellulose ester membrane with a molecular weight cut-off (MWCO) of 2000 Da (Ripley Gold Shanghai Biotechnology Co., Ltd.) for 48 hours (with water changes every 6 hours), and finally subjected to liquid-liquid extraction using dichloromethane (Thermo Fisher Scientific Inc., Cat. No. M501-4) to remove residual organic impurities. The purified CQDs water dispersion was snap-frozen at -20°C in a cryotube before lyophilization, and finally obtained a carbon quantum dot powder.

[0102] Example 2 Preparation of microbial carbon quantum dots ZMNP-CQDs

[0103] This example provides a method for preparing microbial carbon quantum dots ZMNP-CQDs. The difference from Example 1 is that the microbial strain used in step (1) is ZMNP, an ethanologenic Zymomonas mobilis, and the remaining process parameters are the same as those of Example 1.

[0104] Example 3 Preparation of microbial carbon quantum dots PHB-CQDs

[0105] This embodiment provides a method for preparing microbial carbon quantum dots (PHB-CQDs), similar to Example 1, except that the microbial strain used in step (1) is the polyhydroxybutyrate-producing engineered bacterium ZMPt-Flo. N2-EUP All other process parameters are the same as in Example 1.

[0106] Example 4 Microbial carbon quantum dot PHB dopant Preparation of -CQDs

[0107] This embodiment provides a microbial carbon quantum dot PHB. dopant The preparation method of -CQDs is the same as in Example 1, except that step (3) is changed to the following content, and the remaining process parameters are the same as in Example 1: 0.4g of ethanol-producing motile fermentation monocytogenes wild-type ZM4 dry powder and 0.6g of PHB powder are homogenized in 30mL of deionized water for subsequent hydrothermal synthesis process, that is, the proportion of PHB in the total solids is 60% (w / w).

[0108] Example 5: Preparation of microbial carbon quantum dots S-PHB-CQDs

[0109] This embodiment provides a method for preparing microbial carbon quantum dots S-PHB-CQDs, which is the same as in Example 3, except that step (3) is changed to the following content, while the other process parameters are the same as in Example 3: First, 1g ZMPt-Flo N2-EUP The dry powder was reacted with 30 mL of 60% sulfuric acid at 90°C for 20 minutes, and then subjected to secondary carbonization at 200°C for 6 hours in a high-pressure reactor.

[0110] Performance characterization and performance testing

[0111] 1. Morphological characterization of 3 microbial strains and 4 microbial-derived CQDs

[0112] This section describes the characterization of three microbial strains and five microbially derived CQDs involved in the preparation of microbial carbon quantum dots (CQDs) using transmission electron microscopy (Hitachi (China) Ltd., TEM HT7800 120kV), performed by Feynman Biotechnology Co., Ltd. (Wuhan, China). Particle size analysis was performed using ImageJ software to measure the diameter of approximately 100-150 CQDs, and the data were fitted to a log-normal distribution function to calculate the mean diameter and standard deviation. The lattice spacing of the CQDs was measured using Digital Micrograph software. The sample preparation method for the three strains in this example is as follows: 10 ODs were collected... 600nmEach culture unit (equivalent to the volume of a millet grain) was washed by centrifugation in 1.5 mL microtubes and fixed with 2.5% glutaraldehyde (Shanghai Enzyme-Linked Biotechnology Co., Ltd., Catalog No: ML28240-2) at 4°C for 24 hours. Subsequent processing included osmium post-fixation, gradient ethanol dehydration, epoxy resin embedding, ultrathin sectioning using a diamond knife, copper grid loading, uranyl acetate / lead citrate double staining, and finally observation of intracellular components by transmission electron microscopy. Liquid chromatography (HPLC) determination was completed by a 1290 Infinity III liquid chromatograph (Agilent Technologies (China) Co., Ltd.). The PHB content was calculated by replacing the content of crotonic acid with the content of PHB polymer after heating with concentrated sulfuric acid to convert PHB polymer into crotonic acid. The specific steps were as follows: separation was performed using an Aminex HPX-87H ion exchange column (Bio-Rad Laboratories, Inc., USA), detection was performed using an SPD-20A ultraviolet detector (Shimadzu (China) Co., Ltd.), the temperature was 25°C, 5 mmol / L sulfuric acid was used as the mobile phase, and the flow rate was 0.6 mL / min. The initial flow rate was set to 0.2 mL / min during instrument operation, and then gradually increased to 0.5 mL / min at a flow rate of 0.1 mL / min after the column pressure stabilized; the injection volume was 20 μL. Each sample was analyzed in triplicate. Electron transmission microscopy (TEM) was used to characterize the morphology of the three microbial strains, and the results showed that they all presented an elliptical shape with a size of about 0.5-2 μm. Figure 2 In addition, a large amount of PHB was observed in the engineered strain ZMPt-Flo N2 -EUP In addition, a large amount of PHB was observed in the engineered strain ZMPt-Flo N2-EUP The content of PHB in ZMPt-Flo

[0113] Electron transmission microscopy (TEM) was used to characterize the morphology of the five microbial-derived CQDs, and the results showed that the five microbial-derived CQDs presented similar spherical particles with a particle size range of 1.5-4 nm Figure 3 Specifically, the sizes of ZM4-CQDs and ZMNP-CQDs were smaller, at 2.11 ± 0.32 nm and 2.00 ± 0.32 nm, respectively, and the size of PHB dopant -CQDs was 2.73 ± 0.41 nm. Compared with PHB dopant-CQDs exhibited lower crystallinity and higher amorphous carbon dot content. The S-PHB-CQDs particle size was 2.66 ± 0.66 nm with a wide distribution range. This can be due to the strong acidic environment during the hydrothermal process, which destroyed the consistency of carbon core formation, resulting in a wider particle size distribution. High-resolution transmission electron microscopy (HRTEM) images showed that all six CQDs had clear lattice fringes, with an interlayer spacing of 0.19-0.22 nm Figure 3 ), which is consistent with the diffraction plane of graphite carbon. That is, the skeleton of CQDs is composed of a honeycomb structure formed by carbon atoms in a two-dimensional plane, which provides support for the subsequent examples on the chemical structure.

[0114] 2.5 Chemical structure characterization of five microbial-derived CQDs

[0115] This part analyzes the chemical structure characterization of five microbial-derived CQDs. Fourier transform infrared spectroscopy (FTIR) was obtained by potassium bromide (KBr) pellet method (CQDs powder was mixed with potassium bromide according to a mass ratio of 1:100, ground and pressed into a pellet), and recorded in the range of 4000 to 400 cm -1 , with an average scan of 32 times for each CQDs sample. X-ray photoelectron spectroscopy (XPS) analysis was provided by Scientific Compass Wuhan Branch (Hangzhou Research Interest Information Technology Co., Ltd.), and spectral data was deconvoluted and peak-fitted by Avantage software. The morphology of bacterial reaction residues after hydrothermal treatment was observed by scanning electron microscopy (TESCAN GROUP, a.s., SEM Mira Lms).

[0116] The Fourier transform infrared spectroscopy (FT-IR) of five microbial-derived CQDs is shown in Figure 4 (a) figure, which shows that there is an O-H / N-H vibration peak at 3428 cm -1 , a C-H vibration peak at 2932 cm -1 , a C=O vibration peak at 1654 cm -1 , a C=C vibration peak at 1528 cm -1 , a C-N vibration peak at 1385 cm -1 . The stretching vibration of C-O-C and C-O is located at 1121 cm -1 and 1050 cm -1 , respectively. It is worth noting that in S-PHB-CQDs, S=C-C vibration peak at 1143 cm -1 , S=O vibration peak at 1114 cm -1 and 625 cm -1C-S vibration peak. The high content of O-H, N-H, C-O-C and C-O groups on the surface of PHB-CQDs and S-PHB-CQDs indicates that there are abundant carboxyl, amino, carbonyl and ether groups on the surface of these CQDs, which may be the reason for their excellent hydrophilicity, ionic affinity and optical properties. The presence of C, N and O elements in the five kinds of microbial derived CQDs was analyzed by XPS full scan spectrum, and the results are shown in FIGS. (b) and (d) of Figure 4 Compared with ZM4-CQDs (7.10%) and ZMNP-CQDs (11.60%), the N element content in PHB-CQDs (14.49%) is higher. The percentage of O element can reflect the carbonization degree of CQDs, and the exogenous addition of PHB can improve the carbonization degree of CQDs. The percentage of N element can reflect the degree of nitrogen doping in CQDs, and the exogenous addition of PHB can improve the degree of nitrogen doping in CQDs. dopant The percentage of O element in ZM4-CQDs (28.67%). The highest percentage of O element (41.38%) was detected in S-PHB-CQDs, indicating that the acidic environment can further intensify the dehydration condensation and carbonization reaction in the process of CQDs formation. In addition, the sulfur content of 7.61% in S-PHB-CQDs confirms that sulfur is successfully doped into the structure of carbon dots.

[0117] The high-resolution C1s peak XPS spectrum results Figure 5 show that there are C-C / C=C (284.5 eV), C-N (285.1 eV), C-O (286.2 eV) and C=O (287.8 eV) peaks in all CQDs, and an additional C-S peak at 283.5 eV is observed in S-PHB-CQDs. The high-resolution O1s peak XPS spectrum results show that there are C=O, C-OH / C-O-C and S=O Figure 6 The high-resolution N1s peak XPS spectrum results of four kinds of microbial derived CQDs except S-PHB-CQDs are shown in FIGS. (a)-(d) of Figure 7 , and C-N=C, N-(C)3 and N-H groups are observed. In S-PHB-CQDs, specific S=O, C-SO2, C-SO3 and SO4 functional groups are detected Figure 7(Figure (e)). In the synthesis of microbial CQDs, proteins are hydrolyzed into amino acids under heating conditions, and then polymerized and carbonized to form CQDs. The participation of complex biomolecules introduces abundant surface functional groups into CQDs. In addition, the sulfur-containing functional groups on S-PHB-CQDs introduce new energy levels, regulate the band structure, and promote radiative recombination, significantly improving the fluorescence quantum yield of CQDs. XPS and FT-IR spectral analysis results show that the uniform distribution of PHB in the engineered bacteria provides a favorable microenvironment for the synthesis and functional modification of PHB-CQDs. On the one hand, the PHB backbone is composed of ester bonds (-COO-), which usually break down to form monomeric hydroxybutyric acid and its derivatives when treated at 200℃; on the other hand, these degradation products participate in the formation of CQD carbon cores during hydrothermal processes and introduce functional groups such as carboxylic acids, hydroxyl groups, carbonyl groups, ether bonds, and carbon-carbon double bonds on the surface of CQDs.

[0118] Further analysis was conducted using scanning electron microscopy (SEM) to examine the surface roughness and damage level of the hydrothermal synthesis residues, in order to analyze the extent of damage caused by the five microbially derived CQDs during the hydrothermal process. Figure 8 SEM images revealed differences in roughness levels of the residues after the reaction of all types of CQDs. The residue surfaces of ZM4-CQDs, ZMNP-CQDs, and PHB-CQDs exhibited regular and smooth characteristics. Figure 8 Figures (a)-(c) show that biomass decomposition is relatively uniform during hydrothermal synthesis. In contrast, PHB... dopant -CQDs have higher residue roughness than PHB-CQDs, which is attributed to the increased surface irregularity caused by pyrolytic carbon nucleation at the PHB-bacterial interface. Figure 8 (Figure d)). The S-PHB-CQDs residue has the highest roughness, exhibiting a fibrous network porous structure. This complex morphology is the result of the combined effects of sulfuric acid digestion and hydrothermal corrosion. Figure 8 (Figure (e) in the middle).

[0119] The hydrothermal synthesis of CQDs using *Fermentomonas motilityis* biomass as raw material involves a series of steps: First, proteins, polysaccharides, lipids, and nucleic acids in the bacterial cells are hydrolyzed to produce amino acids, monosaccharides, fatty acids, and nucleotides, which serve as carbon precursors for the formation of carbon nuclei. Figure 9 Subsequently, monosaccharides and amino acids undergo dehydration condensation under high temperature and pressure to generate polycyclic aromatic skeletons or nitrogen-doped aromatic structures. These small molecule intermediates further cyclize and dehydrogenate at high temperature to form sp... 2Hybrid carbon network. In this process, the edges of carbon nuclei retain unsaturated dangling bonds (σ bonds or radicals), while the functional groups such as -OH, -NH2, and -COOH produced by the decomposition of bacterial biomass saturate these dangling bonds through covalent anchoring, forming the surface functionalization of CQDs. In the synthesis of PHB-CQDs, the hydrolyzed intracellular PHB derivatives can participate in the aromatization process to form benzene ring structures. At the same time, the depolymerized 3-hydroxybutyric acid monomers undergo hydrolysis, oxidation, and dehydration reactions under hydrothermal conditions to generate short-chain carboxylic acids, unsaturated carboxylic acids, or keto acids. These products cross-link diverse functional groups (-COOH, C-O-C, -OH, C=O) to the surface of CQDs through covalent interactions. The FT-IR and XPS spectral results of ZM4-CQDs and PHB-CQDs show that the increase of the above functional groups in PHB-CQDs further indicates that the oxygen-containing groups produced by the decomposition of intracellular PHB during the hydrothermal synthesis process have been cross-linked Figure 9 ) with each other. In summary, the hydrothermal synthesis process reveals the key differences in the utilization of intracellular precursors and exogenous precursors. For PHB engineering strains, the in situ decomposition of intracellular PHB at high temperature and pressure creates a microenvironment conducive to uniform carbon nucleation and functional group integration. This is in sharp contrast to the exogenous PHB doping method, which leads to irregular carbon nucleus formation and reduced crystallinity, as confirmed by TEM and SEM analysis. This difference highlights the importance of cell structure in guiding the growth of CQDs: the close association of intracellular PHB with bacterial macromolecules such as proteins and lipids promotes synergistic hydrolysis and condensation reactions, while exogenous PHB is hindered from uniform carbonization due to spatial isolation. The present invention reveals that intracellular metabolites can simultaneously serve as carbon sources and "molecular staples" for surface functionalization, providing a blueprint for precise nanomaterial design.

[0120] 3.5 Optical properties and quantum yields of five kinds of microbial-derived CQDs

[0121] This part analyzes the optical properties and quantum yields of five kinds of microbial-derived CQDs. Among them, the ultraviolet-visible absorption spectrum is collected by UV-3600 type ultraviolet-visible spectrophotometer (Shimadzu (China) Co., Ltd.), and the fluorescence emission spectrum, excitation spectrum and fluorescence lifetime spectrum are measured by FluoTime 300 spectrometer (PicoQuant GmbH Company, Germany). The fluorescence lifetime decay curve is fitted by double exponential decay model using FluoFit Pro software (PicoQuant GmbH Company, Germany), and the goodness of fit is evaluated by χ 2 < 1.2. The particle size distribution is analyzed by lognormal fitting (ImageJ) combined with dynamic light scattering (DLS) correlation.

[0122] UV-Vis and downconversion fluorescence spectra of 5 microbial-derived CQDs are as follows: Figure 10 As shown, when excited by ultraviolet light at 351 nm, 375 nm, 387 nm, and 396 nm, respectively, ZM4-CQDs, ZMNP-CQDs, PHB-CQDs, and PHB... dopant - The highest photoluminescence (PL) intensity was observed in these CQDs. Notably, all of these microbial-derived CQDs from *Fermentomonas motilityis* exhibited a significant dependence between emission and excitation wavelengths, characteristic of excitation wavelength-dependent emission behavior. Furthermore, the S-PHB-CQDs exhibited multi-peak emission characteristics, with the main absorption peak located at 380 nm, and only a single peak observed when the excitation wavelength exceeded 300 nm. Figure 10 Figure (e) shows that the CQDs synthesized from *Fermentomonas* strains exhibit significant similarities in light absorption characteristics. Figure 10 The maximum absorption band in the UV region, centered at 270 nm, corresponds to the π-π* electronic transitions of aromatic carbon groups (C=C / C=O). Furthermore, a weak shoulder peak at 320 nm is observed, attributed to the n-π* transitions of surface oxygen-containing functional groups (-OH, -COOH) in all synthesized CQDs. S-PHB-CQDs exhibit significant UV absorption peaks at 240 nm, 260 nm, and 320 nm. The strong oxidizing property of sulfuric acid introduces additional oxygen-containing groups, reducing the core electron cloud density and increasing the transition energy, resulting in a blue shift of the absorption peaks. Additionally, the sulfonic acid groups (S=O) on the aromatic ring induce electron transfer from the carbon core to the sulfonic acid groups, causing a blue shift of the π-π* transition absorption peaks of C=C and C=N groups to 240 nm. The 260 nm absorption peak corresponds to the π-π* transitions of CN and C=O bonds.

[0123] The relative fluorescence quantum yield (QY) of five microbial-derived CQDs is as follows: Figure 11 As shown, the values ​​range from 3.4% to 40.1%. The quantum yields of PHB-CQDs and ZM4-CQDs were 12.9% and 9.7%, respectively, significantly higher in the presence of intracellular PHB material. This suggests that the higher abundance of surface functional groups in PHB-CQDs may more effectively promote radiative recombination. S-PHB-CQDs exhibited the highest fluorescence quantum yield, reaching 40.1%. The presence of sulfur-containing surface functional groups in S-PHB-CQDs effectively reduces the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) band gap, thereby promoting radiative electron-hole recombination through enhanced charge confinement effects. In contrast, PHB... dopantThe fluorescence quantum yield of CQDs was reduced by 64.9% compared to ZM4-CQDs, which was attributed to the local accumulation of PHB outside the bacterial cells during carbonization. Therefore, PHB dopant The carbon core of CQDs can exhibit a decrease in surface functional groups and an increase in amorphous carbon dots formation, which is further supported by the decrease in crystallinity and the presence of a large number of amorphous carbon dots observed in the TEM images in "Performance Tests and Performance Characterization 1."

[0124] The fluorescence lifetime spectra of 5 microbe-derived CQDs are shown in Figure 12 The surface state lifetime of S-PHB-CQDs is the highest (τ1), indicating that sulfur doping increases the proportion of surface state luminescence. Although the surface state and core state lifetimes of S-PHB-CQDs are both shortened, the fluorescence quantum yield is increased. This indicates an increase in radiative recombination efficiency and a decrease in non-radiative recombination processes.

[0125] 4. Metal ion detection of microbe-derived CQDs

[0126] The fluorescence quenching mechanism between CQDs and Fe 3+ is mainly affected by the combined effects of inner filter effect (IFE), static quenching, and dynamic quenching, with IFE playing a dominant role. However, at low Fe 3+ concentrations, microbe CQDs rich in oxygen-containing functional groups (-COOH, C-O-C, -OH, C=O) on the surface are prone to form coordinate bonds ( Figure 13 ) with Fe 3+ . The transfer of electrons from oxygen-containing functional groups to Fe 3+ results in the formation of non-fluorescent complexes. In "Performance Tests and Performance Characterization 3." of the present invention, it has been successfully demonstrated that PHB-CQDs and S-PHB-CQDs are rich in polar amino groups and oxygen-containing functional groups on the surface compared to other microbe CQDs. Therefore, the synthetic PHB-CQDs prepared in the present invention exhibit potential as Fe 3+ fluorescent probes.

[0127] In the first aspect, 13 metal ions were selected to investigate their effects on the fluorescence intensity of PHB-CQDs under 350 nm excitation, specifically:

[0128] (1) Different metal ions were dissolved in phosphate buffered saline (PBS, Thermo Fisher Scientific Inc., Cat. No. 20012027) and diluted, then mixed with 1 mg / mL PHB-CQDs solution at a volume ratio of 1:1, so that the final concentration of metal ions was 5 mmol / L and the concentration of PHB-CQDs was 0.5 mg / L. The fluorescence spectrum was recorded (the determination method is the same as that in the "Performance Test and Performance Characterization 3." of the present application, the same below), the normalized fluorescence intensity was calculated, and the fluorescence quenching efficiency of 13 kinds of metal ions on PHB-CQDs was determined.

[0129] (2) The concentration of 13 kinds of metal ions was adjusted to a final concentration of 1 mmol / L, the fluorescence spectrum was recorded, the normalized fluorescence intensity was calculated, and the interference of other metal ions on Fe 3+ detection was evaluated.

[0130] (3) PHB-CQDs were diluted to 0.125 mg / mL, and the fluorescence quenching effect of PHB-CQDs on different concentrations of Fe 3+ was tested, and the related fluorescence intensity data were recorded for subsequent experiments.

[0131] The results showed that the addition of Fe 3+ caused almost complete fluorescence quenching of PHB-CQDs, while the quenching effect of other metal ions (Hg 2+ and Cu 2+ ) was negligible ( Figure 14 (a) of the figure). The effect of Fe 3+ coexisting with other metal ions on the fluorescence quenching of PHB-CQDs showed that the interference of competitive ions was minimal ( Figure 14 (b) of the figure). Further results showed that after adding Fe 3+ (0-5 mmol / L) to 1 mg / mL PHB-CQDs solution, the fluorescence was gradually quenched ( Figure 14 (c) of the figure).

[0132] In the second aspect, the linear relationship of ZM4-CQDs, ZMNP-CQDs, PHB-CQDs and PHB dopant -CQDs to Fe 3+ quenching was further tested, and the stability of the five kinds of microbial derived CQDs at different pH was tested. Specifically:

[0133] (1) Using the fluorescence quenching effect test data of PHB-CQDs on different concentrations of Fe 3+ , the normalized quenching ratio (F0-F) / F0 was calculated by Origin 2024 software (OriginLab Corporation, America) through the least square method, and the linear relationship of PHB-CQDs to Fe3+ The concentration (pmol / L) was linearly regressed to draw a calibration curve, and the regression equation was obtained. The goodness of fit was evaluated by the determination coefficient (R 2 ).

[0134] (2) Based on the signal-to-noise ratio (S / N) method, the detection limit (LOD) of the five kinds of microbial-derived CQDs as fluorescent probes was evaluated and calculated.

[0135] (3) 37% hydrochloric acid and 10% sodium hydroxide solution were used to prepare solutions with pH values of 0-14 (integers). Fe 3+ was added to the solution until the concentration of Fe 3+ was 50 pmol / L. CQDs were diluted to 0.125 mg / mL, the normalized fluorescence intensity was calculated, and the stability of the five kinds of microbial-derived CQDs at different pH values was evaluated.

[0136] The results showed that the fluorescence intensity of ZM4-CQDs, ZMNP-CQDs, PHB-CQDs and PHB dopant -CQDs all decreased with the increase of the concentration of Fe 3+ solution ( Figure 15 ). Among them, PHB-CQDs had the highest sensitivity to Fe 3+ , and the linear range was 2.5-100 pmol / L (R 2 = 0.998). Compared with PHB-CQDs, the linear detection ranges of ZM4-CQDs, ZMNP-CQDs and PHB dopant -CQDs were 20-75 pmol / L (R 2 = 0.994), 20-75 pmol / L (R 2 = 0.977) and 10-100 pmol / L (R 2 = 0.998), respectively. Through the pH fluorescence stability test of CQDs ( Figure 16 ), it was found that the four kinds of microbial-derived CQDs except S-PHB-CQDs remained good fluorescence stability in the pH range of 3-11. However, in a strong acidic or alkaline environment, the fluorescence intensity of microbial-derived CQDs decreased significantly. In view of the large amount of strong acidic wastewater existing at present, the present application expands the pH range of S-PHB-CQDs for Fe 3+ detection, which still maintains good fluorescence stability in the pH range of 0-3 ( Figure 16 ). Further test of the linear relationship of S-PHB-CQDs to Fe 3+ quenching under the condition of pH=0 showed that the linear detection range was 5-100 pmol / L (R 2 = 0.999) ( Figure 15 ).

[0137] The detection limits (LOD) of ZM4-CQDs, ZMNP-CQDs, PHB-CQDs, PHB dopant -CQDs and S-PHB-CQDs were 7.16 μmol / L, 7.33 μmol / L, 0.507 μmol / L, 2.31 μmol / L and 0.997 μmol / L, respectively. The results showed that the introduction of intracellular and extracellular materials could enhance the detection ability of CQDs for Fe 3+ , and the intracellular materials played a more significant role. Notably, PHB-CQDs had the lowest LOD, indicating its excellent sensitivity for the quantification of trace Fe 3+ . In addition, S-PHB-CQDs still maintained excellent detection sensitivity and high quantum yield characteristics in a strongly acidic environment.

[0138] Example 6 Application of Microbial-Derived CQDs in the Detection of Fe3+ in Water Quality Samples

[0139] To verify the practical application potential of the quenching-type fluorescent PHB-CQDs and S-PHB-CQDs sensors for the detection of Fe 3+ , this example conducted comprehensive spiked recovery tests using samples 1-5 (covering tap water, lake water, industrial wastewater, iron corrosion wastewater, etc.) (Table 1). The specific source information of the water quality samples is as follows: water sample 1 was a hydrochloric acid solution with pH = 0; water sample 2 was a domestic tap water collected in Wuhan City, Hubei Province; water sample 3 was a lake water collected in Sha Lake, Wuhan City, Hubei Province; water sample 4 was an industrial wastewater collected in Wuhan Iron and Steel Co., Ltd.; and water sample 5 was an iron corrosion wastewater collected in Wuhan City, Hubei Province. Among them, the prepared S-PHB-CQDs were used to evaluate the Fe 3+ content in water sample 1, while PHB-CQDs were used to evaluate the Fe 3+ content in water samples 2-5.

[0140] Table 1: Detection of Fe3+ in actual samples by PHB-CQDs and S-PHB-CQDs 3+

[0141]

[0142] According to the analysis and determination by inductively coupled plasma optical emission spectrometry (ICP-OES), the initial Fe 3+ concentrations of samples 1-3 were 0 μmol / L, 0 μmol / L and 0.3 μmol / L, respectively, and one was below the detection limit. Subsequently, the responses of PHB-CQDs and S-PHB-CQDs to Fe 3+ in the samples were tested. Sample 4 was not additionally added with Fe 3+At that time, its Fe 3+ The concentration had already reached 1.6 μmol / L, exceeding the linear detection range of PHB-CQDs. Therefore, in calculating the detection range of Fe by PHB-CQDs... 3+ When the recovery rate was considered, this sample was not included. Samples 2-5 were analyzed for Fe by PHB-CQDs. 3+ The recoveries ranged from 96.3% to 106.8% (Table 1), confirming the suitability of the fluorescent PHB-CQDs sensor for detecting Fe. 3+ The sensor exhibits practical reliability. Furthermore, its stability was further investigated under extreme acidic conditions (Sample 1, pH = 0, HCl matrix). Notably, the S-PHB-CQDs maintained high detection accuracy, with recoveries ranging from 102.5% to 106.8%, and extremely high precision (relative standard deviation RSD < 0.8%). This performance indicates that the sensor is suitable for industrial applications involving strongly acidic matrices.

[0143] Example 7: PHB-CQDs used to prepare Fe 3+ Detection paper chip

[0144] This embodiment provides a Fe 3+ The detection paper chip, used for on-site semi-quantitative detection of ferric ions in water samples, is prepared by the following steps:

[0145] (1) Dissolve 0.4g sodium carboxymethyl cellulose in 10mL of 100℃ deionized water, add 3mL of 1% polyvinyl alcohol and 2mL of 10% citric acid solution, and cool to 40-45℃ to obtain a paper-based carrier.

[0146] (2) PHB-CQDs were added to a paper-based support at 40-45℃ to obtain Fe 3+ Detect paper chips.

[0147] Specifically, the paper-based detection device needs to be used under 350nm ultraviolet light excitation.

[0148] This paper-based detection device uses a paper chip to detect Fe. 3+ For semi-quantitative detection, PHB-CQDs immobilized within a paper chip exhibited strong blue fluorescence under 350nm UV light. With the detection of Fe... 3+ As the concentration increases (from 0 to 2 mmol / L), the fluorescence gradually quenches until it is completely extinguished. Figure 17 ).

[0149] Compared with wild-type strain ZM4-CQDs, PHB-CQDs have Fe 3+The detection sensitivity was improved by 14 times (performance characterization and performance testing 4.), highlighting the crucial role of intracellular biopolymers in determining surface chemical properties. The spiked recovery test in actual water samples in Example 6 of this invention demonstrated excellent performance, validating its reliability in complex matrices. Furthermore, S-PHB-CQDs achieved an unprecedented quantum yield of 40.1%, setting a new benchmark for microbial-derived fluorescent nanomaterials. Even under extremely acidic conditions (pH=0), S-PHB-CQDs maintained stable Fe... 3+ Detection sensitivity is crucial for the analysis of industrial wastewater in industries such as mining, metallurgy, and chemicals.

[0150] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of carbon quantum dots in the detection of ferric ions in water samples, characterized in that, The carbon quantum dots are PHB-CQDs or S-PHB-CQDs; The preparation method of carbon quantum dots PHB-CQDs includes the following steps: SA-1, activated and expanded polyhydroxybutyrate-producing engineered bacteria ZMPt-Flo N2-EUP strain ZMPt-Flo was obtained. N2-EUP bacterial solution; SA-2, strain ZMPt-Flo N2-EUP After centrifugation, washing, and freeze-drying, the bacterial culture was used to obtain strain ZMPt-Flo. N2-EUP Dry powder; SA-3, 1 g of strain ZMPt-Flo N2-EUP The dry powder was homogenized with deionized water at a mass-volume ratio of 1 g: 30 mL and then placed in a reaction vessel for hydrothermal reaction at 200 °C for 6 h. The preparation method of carbon quantum dots S-PHB-CQDs includes the following steps: SB-1, activated and expanded polyhydroxybutyrate-producing engineered bacteria ZMPt-Flo N2-EUP strain ZMPt-Flo was obtained. N2-EUP bacterial solution; SB-2, strain ZMPt-Flo N2-EUP After centrifugation, washing, and freeze-drying, the bacterial culture was used to obtain strain ZMPt-Flo. N2-EUP Dry powder; SB-3, 1 g of strain ZMPt-Flo N2-EUP The dry powder was reacted with 30 mL of 60% sulfuric acid at 90 °C for 20 minutes, and then placed in a reaction vessel for secondary carbonization at 200 °C for 6 hours. When the carbon quantum dots are PHB-CQDs, the pH range of the water sample is 3-11; When the carbon quantum dots are S-PHB-CQDs, the pH range of the water sample is 0-3.

2. The application as described in claim 1, characterized in that, Includes the following steps: SC-1. Premix the water sample with the carbon quantum dots to obtain a sample premix solution; SC-2, determination of Fe-free 3+ The fluorescence intensity F0 of the carbon quantum dot solution at the maximum ultraviolet excitation wavelength; SC-3. Determine the fluorescence intensity F of the sample premix described in step SC-1 at the maximum ultraviolet excitation wavelength; SC-4, based on the normalized quenching ratio (F0-F) / F0 and Fe 3+ Concentration calibration curve equation, calculate Fe 3+ Concentration, of which Fe 3+ The unit of concentration is μmol / L.

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