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CN-121978067-A - Optical probe trace substance detection method based on quantum dot fluorescence

CN121978067ACN 121978067 ACN121978067 ACN 121978067ACN-121978067-A

Abstract

The invention relates to the technical field of analytical chemistry and nano biological sensing, and discloses a method for detecting trace substances by using an optical probe based on quantum dot fluorescence. According to the method, core-shell quantum dots with surface modification specific recognition units are used as probes, a fluorescence lifetime attenuation curve and a full-band emission spectrum are synchronously acquired under pulse excitation, and the average fluorescence lifetime and spectrum peak position change are analyzed in a combined mode through a multiparameter decoupling model, static quenching, dynamic quenching and mixed quenching mechanisms are distinguished, so that the existence and concentration of a target object are accurately judged. The system comprises a pulse ultraviolet light source, a time-dependent single photon counting module and a high-resolution grating spectroscopic detector array. The invention obviously improves the selectivity, sensitivity and anti-interference capability of detection through the two-dimensional feature fusion, and the detection limit reaches picomolar per upgrade, thereby being applicable to the high-reliability detection of trace substances in environmental, food and clinical samples.

Inventors

  • ZHAO CHUNXIANG
  • WANG XUEQIN
  • WANG JINLEI
  • DING YONGJIE

Assignees

  • 周口师范学院

Dates

Publication Date
20260505
Application Date
20260122

Claims (10)

  1. 1. The method for detecting the trace substance by using the optical probe based on quantum dot fluorescence is characterized by comprising the following steps of: Constructing a core-shell quantum dot optical probe with a fixed surface ligand structure, and modifying a molecular recognition unit with high affinity and specific recognition capability on the surface of the core-shell quantum dot optical probe; Mixing and incubating a sample solution to be detected with the quantum dot optical probe to enable a target substance to interact with the probe in a specific way; Adopting a pulse ultraviolet excitation light source to irradiate the reaction system, and synchronously collecting a fluorescence lifetime attenuation curve and a full-band emission spectrum of the quantum dot probe; Based on a preset multiparameter decoupling model, carrying out joint analysis on fluorescence lifetime parameters and emission spectrum characteristics, judging the type of a quenching mechanism and outputting a quantitative concentration value of a target substance.
  2. 2. The quantum dot fluorescence-based optical probe trace substance detection method according to claim 1, wherein a core-shell quantum dot optical probe having a fixed surface ligand structure is constructed, and a molecular recognition unit having high affinity and specific recognition ability for a target trace substance is modified on the surface thereof, comprising: Preparing quantum dots of a cadmium sulfide/zinc selenide core-shell structure, wherein the core diameter is 3-5 nanometers, and the shell thickness is 1-2 nanometers; Carrying out water phase transfer treatment on the quantum dots through mercaptopropionic acid to expose carboxyl functional sites on the surfaces of the quantum dots; and covalently connecting the molecular recognition unit to the carboxyl through carbodiimide coupling reaction to form a stable biological or chemical recognition interface.
  3. 3. The method for detecting the trace substance based on the optical probe of the quantum dot fluorescence according to claim 2, wherein the molecular recognition units are monoclonal antibodies, nucleic acid aptamers or molecularly imprinted polymers, and the number of the molecular recognition units loaded on the surface of each quantum dot is 5 to 15.
  4. 4. The method for detecting trace substances by using a quantum dot fluorescence-based optical probe according to claim 1, wherein the step of mixing and incubating a sample solution to be detected with the quantum dot optical probe comprises the steps of: Centrifuging a sample solution to be tested to remove particles, adjusting the pH value to 7.0+/-0.2, and adding ethylenediamine tetraacetic acid as a masking agent; and mixing the pretreated sample solution with the quantum dot optical probe according to the volume ratio of 1:1, and incubating for 30 minutes at the constant temperature of 37 ℃ in a dark place.
  5. 5. The method for detecting trace substances by using optical probes based on quantum dot fluorescence according to claim 1, wherein the method for synchronously collecting a fluorescence lifetime decay curve and a full-band emission spectrum of the quantum dot probes by irradiating a reaction system by using a pulse ultraviolet excitation light source comprises the following steps: a pulse ultraviolet light emitting diode with peak wavelength of 371 nanometers, pulse width of 10 nanoseconds and repetition frequency of 1000 hertz is used as an excitation light source; recording a fluorescence lifetime decay histogram with a time resolution accuracy of not less than 50 picoseconds by a time-dependent single photon counting module; the complete emission spectrum profile of the 400-700 nanometer wave band is synchronously acquired by the grating spectroscope array with the spectrum resolution of 0.5 nanometer.
  6. 6. The method for detecting trace substances of optical probes based on quantum dot fluorescence according to claim 5, wherein the time-dependent single photon counting module shares the same excitation timing reference with the grating spectroscopic detector array, ensuring that fluorescence lifetime data and emission spectrum data are strictly synchronized in time.
  7. 7. The method for detecting trace substances of an optical probe based on quantum dot fluorescence according to claim 1, wherein the combined analysis of fluorescence lifetime parameters and emission spectrum characteristics based on a preset multiparameter decoupling model, the determination of the type of quenching mechanism and the output of a quantitative concentration value of a target substance, comprises: A double-exponential decay model is adopted for the fluorescence lifetime decay curve Fitting and calculating the average fluorescence lifetime ; Extracting peak wavelengths from the emission spectrum Full width at half maximum FWHM and spectral centroid ; Will be 、 、 、 、 、FWHM、 And inputting the target substance into the multi-parameter decoupling model as an input characteristic, and outputting a quantitative concentration value of the target substance.
  8. 8. The method for detecting trace species in an optical probe based on quantum dot fluorescence according to claim 7 wherein determining the type of quenching mechanism comprises: If it is A relative rate of change of greater than 10 percent The offset is less than 0.5 nanometers, and the dynamic quenching is judged; If it is A rate of change of less than 5 percent The offset is greater than 1.5 nanometers, and the static quenching is judged; If it is Shortened and Synchronization shift, a hybrid quenching mechanism is determined.
  9. 9. The method for detecting trace substances of optical probes based on quantum dot fluorescence according to claim 7, wherein the multi-parameter decoupling model is established through standard sample calibration; the standard sample comprises a target trace substance solution with known concentration and a plurality of interference substance solutions; measuring the standard samples of each type 、 、 、 、 、FWHM、 And training a gradient lifting decision tree model or a support vector regression model by taking the real concentration as a label.
  10. 10. The method for detecting trace species of an optical probe based on quantum dot fluorescence according to claim 9, wherein the concentration of the target trace species in the standard sample ranges from 10 picomoles per liter to 100 nanomoles per liter; The model training adopts five-fold cross validation, input characteristics are standardized, and output concentration values adopt logarithmic scale coding.

Description

Optical probe trace substance detection method based on quantum dot fluorescence Technical Field The invention belongs to the technical field of analytical chemistry and nano biological sensing, and particularly relates to a method for detecting trace substances by using an optical probe based on quantum dot fluorescence. Background With the deep application of trace substance detection in the fields of biomedicine, environmental monitoring, food safety and the like, a high-sensitivity analysis method based on a fluorescent probe is widely focused. The quantum dot is an ideal material for constructing a fluorescent probe due to wide excitation and narrow emission, high quantum yield and excellent light stability. However, conventional detection strategies based on fluorescence intensity variation face fundamental limitations in complex biological environments in that when a target is bound to a probe to induce fluorescence quenching, the signal variation cannot effectively distinguish whether it is due to a specific recognition reaction or due to interference factors such as non-specific adsorption, micro-environmental disturbance (e.g., pH fluctuation, ionic strength variation) or photobleaching. The ambiguity of the signal source severely restricts the reliability and quantitative accuracy of the detection result, and is particularly prominent in dynamic heterogeneous systems such as living cells. The fluorescent lifetime imaging technology based on quantum dots provides a new path for breaking through the bottleneck. The fluorescence lifetime is taken as a molecular intrinsic property to reflect the relaxation dynamics process of the excited state carrier, and the value of the fluorescence lifetime mainly depends on the interaction mechanism of the probe and the local microenvironment and is insensitive to the intensity dependence factors such as the probe concentration, the excitation light intensity, the optical path loss and the like. By accurately measuring the fluorescence lifetime change of the quantum dot in the presence of the target, a kinetic fingerprint directly associated with a specific molecular recognition event can be theoretically established, thereby avoiding the inherent defect of intensity detection. While the prior art has attempted to use time-dependent single photon counting (TCSPC) for fluorescence lifetime measurement, multiple challenges are faced in the trace species detection scenario, on the one hand, the conventional system lacks the space-time resolution capability for weak signals in living cells, and is difficult to realize rapid and low phototoxicity dynamic tracking on a subcellular scale, and on the other hand, the existing platform does not deeply couple lifetime information with a specific recognition mechanism, fails to construct a quantitative mapping model between lifetime difference and target species concentration, and does not effectively utilize lifetime parameters to suppress non-specific background interference. Therefore, a novel detection paradigm integrating quantum dot probe design, fluorescence lifetime imaging and time-resolved signal processing is needed to realize high-specificity, high-sensitivity and dynamically-resolvable trace substance detection in a living environment. Disclosure of Invention The invention provides a method for detecting trace substances of an optical probe based on quantum dot fluorescence, which aims to solve the technical problem that the traditional fluorescence intensity detection technology cannot distinguish fluorescence quenching sources. In the prior art, when a target analyte interacts with a quantum dot photoprobe, the decrease in fluorescence signal is generally directly attributed to the presence of the target, however, this fluorescence quenching phenomenon may be caused by a variety of physical or chemical factors including, but not limited to, environmental temperature fluctuations, solvent polarity changes, photobleaching effects, non-specific adsorption interference, and dynamic quenching of coexisting ions. Because only single fluorescence intensity is used as a criterion, the false positive or false negative response caused by the non-target factors is difficult to be effectively eliminated by the existing method, so that the accuracy, selectivity and reliability of trace substance detection are severely restricted. In order to overcome the defects, the invention provides a quantum dot optical probe detection method integrating fluorescence lifetime and emission spectrum two-dimensional characteristic analysis. The method comprises the steps of constructing a core-shell type quantum dot with a fixed surface ligand structure as a basic fluorescent probe, modifying a molecular recognition unit with high affinity and specific recognition capability for a target trace substance on the surface of the basic fluorescent probe, synchronously collecting a fluorescence lifetime attenuation curve and a full-band emissi