Label-free fluorescent method for early detection of childhood tumors

Abstract

The application belongs to the technical field of disease diagnosis, and particularly relates to a method for label-free fluorescent early detection of tumors in children. In order to construct a non-invasive early diagnosis method for neuroblastoma, the application finds through research that the fluorescence intensity of plasma of tumor patients is significantly reduced under 280 nm excitation, and tryptophan (TRP) is the main source of the difference fluorescence through metabolomics verification. It is also found that the fluorescence lifetime of flavin / lipofuscin of patients in the late stage is significantly prolonged. The TRP fluorescence intensity and the flavin / lipofuscin fluorescence lifetime parameters are combined to build a model, and the ROC analysis result shows that the AUC of the combination for distinguishing normal people from patients in early and late stages and early and late stages is 0.77, 0.92 and 0.99 respectively. The detection method does not need fluorescent labeling, is simple and convenient to operate, low in cost, excellent in diagnosis performance, suitable for screening of children patients, and can provide core optical parameters for research and development of portable diagnostic equipment, and has good clinical popularization value.

Description

Technical Field

[0001] This invention belongs to the field of disease diagnosis technology, specifically relating to a method for early detection of childhood tumors using label-free fluorescence. Background Technology

[0002] Neuroblastoma, a malignant solid tumor originating from primitive neural crest cells of the neuroectoderm, accounts for 8%-10% of all childhood cancers and has a mortality rate of approximately 15%, making it one of the leading causes of death in children. This tumor exhibits significant clinical heterogeneity, with course ranging from spontaneous regression to localized lesions or widespread metastasis. Early diagnosis of neuroblastoma is extremely challenging due to its often occult primary sites, high recurrence rate, and significant tendency for distant metastasis. At clinical diagnosis, approximately 50% of patients already have metastatic disease. Although multimodal treatments (including surgery, radiotherapy, chemotherapy, autologous stem cell transplantation, and immunotherapy) have significantly improved efficacy over the past few decades, the recurrence rate for high-risk neuroblastoma patients remains above 50%, and the 5-year survival rate is still less than 50%. Long-term survival of neuroblastoma decreases significantly with increasing age at diagnosis. Therefore, there is an urgent need to establish reliable early diagnostic techniques to improve the early detection rate of neuroblastoma and provide crucial guidance for patient treatment planning and prognosis prediction.

[0003] Currently, early diagnosis and risk assessment of neuroblastoma in children can be conducted using a combination of auxiliary examinations, including laboratory tests, histopathological examinations, gene sequencing, and imaging techniques. Serological tests are the simplest and most convenient method; for example, measuring serum ferrous sulfate (SF), serum lactate dehydrogenase (LDH), and neuron-specific enol (NSE) levels is significant for the diagnosis and prognosis of neuroblastoma. However, the specificity and sensitivity of laboratory tests vary greatly, and to date, characteristic biochemical indicators for early diagnosis and prognostic assessment of neuroblastoma remain to be determined. Histopathological examination and gene sequencing are commonly used methods in clinical laboratories to determine tumor pathology and detect tumor markers, but histopathological examination has drawbacks such as cumbersome procedures, low sensitivity, and poor reproducibility; while gene sequencing for biomarker screening is tedious, time-consuming, and expensive. Currently, imaging techniques used clinically for the diagnosis of neuroblastoma include computed tomography (CT) and magnetic resonance imaging (MRI). 123 I-labeled m-iodobenzylguanidine single-photon emission and X-ray computed tomography (SPECT) 123 I-MIBG SPECT-CT and 2-[18F]-fluoro-2-deoxy-D-glucose-positron emission tomography / computed tomography (CET-CT) 18CT and MRI (F-FDG PET-CT, etc.) have low sensitivity and specificity in detecting lesions, especially in the identification of early cancerous changes in tumors. 123 I-MIBG SPECT-CT and 18 F-FDG PET-CT are two nuclear medicine imaging methods recommended by clinical guidelines, exhibiting high sensitivity and specificity in diagnosing neuroblastoma staging and metastatic lesions. However, due to the inherent risks associated with contrast agents, radiation exposure, and high cost, this type of metabolic imaging is not suitable for screening, routine monitoring, or early detection. The high heterogeneity of neuroblastoma makes accurate detection of early lesions difficult, and the use of exogenous probes can easily interfere with metabolic changes, thus affecting the assessment of early cancerous transformation. Therefore, there is an urgent need to develop a diagnostic technique that is sensitive to functional metabolic changes and requires no labeling. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides a combination of optical detection parameters (TRP fluorescence intensity and flavin / lipofuscin fluorescence lifetime) for the diagnosis of neuroblastoma in children. This combination can enable early auxiliary diagnosis and risk assessment of childhood tumors (neuroblastoma), and can also provide core optical parameters for the development of portable diagnostic devices, thus possessing good clinical application value.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides an application of a combination of optical detection parameters in the preparation of diagnostic equipment or procedures for neuroblastoma in children. The combination of optical detection parameters comprises: the fluorescence intensity of tryptophan in body fluids detected at a 280 nm excitation wavelength, and the fluorescence lifetime fitting component of flavin / lipofuscin in body fluids detected at a 450 nm excitation wavelength. .

[0006] These are three types of characteristic fluorescence lifetime components obtained by fitting a three-exponential fluorescence decay model: short, medium, and long. They correspond to the different fluorescence decay kinetics of flavin and lipofuscin in body fluids (such as plasma) in free, weakly protein-bound, and strongly protein-bound states. The three components characterize different molecular microenvironment states and together reflect the changes in the existence form of endogenous fluorescent metabolites during tumor development.

[0007] Preferably, the body fluid includes plasma, serum, urine, or cerebrospinal fluid. More preferably, the body fluid is plasma.

[0008] Preferably, the diagnostic device or procedure for pediatric neuroblastoma can be used for the early diagnosis of neuroblastoma or to differentiate between early and late-stage neuroblastoma; wherein, compared with the normal population, the autofluorescence intensity of body fluids from patients with early and late-stage tumors is significantly reduced under 280 nm excitation, and the signal is relatively weaker in early-stage patients; the fitted component of the flavin / lipofuscin fluorescence lifetime in the body fluids of patients with late-stage tumors... The rate was significantly higher than that of patients with early-stage cancer.

[0009] Preferably, the diagnostic testing equipment includes a fluorescence spectrophotometer and a fluorescence lifetime spectrometer equipped with a TCSPC module.

[0010] More preferably, fluorescence lifetime fitting component The acquisition method is as follows: using a fluorescence lifetime spectrometer equipped with a TCSPC module, the detection time is set to 50 ns and the number of channels to 1024. Acquisition is stopped when the peak count reaches 7000. Then, the excitation wavelength is set to 450 nm, and the emission signals of flavin / lipofuscin are acquired in the 525~575 nm channel. Finally, a triple exponential decay model is used. The fluorescence lifetime parameters are obtained by fitting the decay curve. Specifically, the fluorescence lifetime decay curve of bodily fluids (such as blood plasma) is first measured on a fluorescence lifetime spectrometer. Then, the software applies a triple exponential decay model to fit the curve, and automatically calculates and outputs three characteristic fluorescence lifetime parameters. .

[0011] More preferably, the method for obtaining the fluorescence intensity of tryptophan in body fluids is as follows: the body fluid sample is subjected to autofluorescence spectroscopy detection using a fluorescence spectrophotometer, the excitation wavelength is set to 280 nm, and the excitation bandwidth and emission bandwidth are both 2.5 nm, and then the autofluorescence intensity corresponding to tryptophan in body fluids is measured.

[0012] More preferably, the PBS blank background spectrum is measured simultaneously during the detection process, and background interference is subtracted.

[0013] A second aspect of the present invention also provides a non-invasive early detection method for neuroblastoma based on plasma optical characteristics, the method specifically comprising the following steps: S1. The autofluorescence spectrum of plasma samples was detected by a fluorescence spectrophotometer. The excitation wavelength was set to 280 nm, and the autofluorescence intensity corresponding to plasma tryptophan was measured. S2. A fluorescence lifetime spectrometer equipped with a TCSPC module was used to detect the fluorescence lifetime of plasma samples. An excitation wavelength of 450 nm was set, and the emission signals of flavin / lipofuscin were acquired in the 525–575 nm channel. A triple exponential decay model was then used. Fluorescence lifetime parameters of flavin / lipofuscin were obtained by fitting decay curves. ; S3. Measure the fluorescence intensity of tryptophan and the fluorescence lifetime parameters of flavin / lipofuscin under 280 nm excitation. They are used together as input variables to construct a logistic regression diagnostic model; S4. The logistic regression diagnostic model is used to differentiate between normal individuals, early-stage neuroblastoma patients, and late-stage neuroblastoma patients.

[0014] Preferably, the criteria for the logistic regression diagnostic model are as follows: the AUC for distinguishing between normal individuals and early-stage neuroblastoma is 0.77; the AUC for distinguishing between normal individuals and late-stage neuroblastoma is 0.92; and the AUC for distinguishing between early-stage and late-stage neuroblastoma is 0.99.

[0015] Compared with the prior art, the beneficial effects of the present invention are: To construct a non-invasive early diagnostic method for neuroblastoma, this invention sequentially conducted autofluorescence spectroscopy detection, amino acid-targeted metabolomics analysis, fluorescence lifetime detection, and component analysis, combined with logistic regression to build a diagnostic model. Fluorescence spectroscopy results showed a significant decrease in plasma fluorescence intensity in tumor patients under 280 nm excitation, and metabolomics verification confirmed that tryptophan (TRP) was the main source of this differential fluorescence. Fluorescence lifetime detection showed a significantly prolonged flavin / lipofuscin fluorescence lifetime in advanced patients, while porphyrin fluorescence lifetime showed no significant difference. Modeling a combination of TRP fluorescence intensity and flavin / lipofuscin fluorescence lifetime parameters, ROC analysis showed that the AUCs for distinguishing normal individuals from early and late-stage patients, and for early and late stages, were 0.77, 0.92, and 0.99, respectively. This detection method requires no fluorescent labeling, is simple to operate, low in cost, and has excellent diagnostic performance. It is not only suitable for screening pediatric patients but can also provide core optical parameters for the development of portable diagnostic devices, possessing good clinical application value.

[0016] Specifically, compared with existing technologies such as serological testing, histopathology, gene sequencing, and imaging techniques such as CT / MR / SPECT / PET, this invention has the following significant advantages: (1) It achieves label-free and non-invasive early diagnosis: only a small amount of plasma is needed, no exogenous fluorescent probes or contrast agents are required, radiation exposure and metabolic interference are avoided, and it is suitable for screening and routine monitoring of pediatric patients.

[0017] (2) Significantly distinguishing normal from early neuroblastoma: The fluorescence intensity of tryptophan (TRP) was compared with the fluorescence lifetime of flavin / lipofuscin (FRP). The combination of parameters achieved an AUC of 0.77 when distinguishing between normal controls and early-stage patients using a logistic regression model, indicating that the combined parameters have a good ability to identify early lesions.

[0018] (3) Highly effective in distinguishing between normal and late-stage patients: The AUC of 0.92 for distinguishing between normal controls and late-stage patients with the same combination of parameters demonstrates excellent diagnostic performance.

[0019] (4) Accurately distinguish between early and late-stage patients: The AUC for distinguishing between early and late-stage neuroblastoma reached 0.99, suggesting that this technology can not only be used for early screening, but also assist in disease staging and progression assessment.

[0020] (5) The metabolic mechanism is clear and the results are explainable: Through amino acid-targeted metabolomics, it was confirmed that TRP is the main contributor to the difference in plasma fluorescence intensity under 280 nm excitation, and its trend is completely consistent with the fluorescence intensity; the fluorescence lifetime of flavin / lipofuscin is significantly prolonged in the late stage, providing a clear metabolic basis for diagnosis.

[0021] (6) Simple, fast and low cost: The fluorescence spectroscopy and fluorescence lifetime detection process is simple and does not require complicated sample pretreatment (such as derivatization, amplification, etc.). The detection cost is much lower than existing technologies such as gene sequencing and PET-CT, and it is easy to promote clinical application and large-scale screening.

[0022] (7) Provides core parameters for subsequent equipment development: The selected optical parameters (TRP fluorescence intensity under 280 nm excitation; three fluorescence lifetime components of flavin / lipofuscin under 450 nm excitation) can be used as key indicators for dedicated detection equipment, supporting the development of portable diagnostic instruments. Attached Figure Description

[0023] Figure 1 The results are from plasma autofluorescence spectroscopy detection; among them, Figure 1 Fluorescence spectra of A-1J excited at wavelengths of 280, 300, 330, 370, 390, 410, 460, 490, 520, and 561 nm, respectively; Figure 1 K is Figure 1 Quantitative analysis of fluorescence intensity from A-1J fluorescence spectrum; experimental data are presented in the form of... ±SD represents the number of biological replicates n = 58 in the control group, n = 11 in the early group, and n = 41 in the advanced group. This indicates a comparison with the normal group (Control). <0.05, <0.01 and <0.001; # This indicates a comparison with the early stage. # P<0.05, ## P<0.01 and ### P<0.001.

[0024] Figure 2-4 For amino acid-targeted metabolite profiling analysis; among which, Figure 2 Total ion chromatogram (TIC) of mixed standard samples; Figure 3 Total ion chromatogram (TIC) of clinical specimens; Figure 4 For the relative quantitative analysis of tryptophan (TRP), tyrosine (TYR), and phenylalanine (PHE) in plasma; experimental data are presented as follows: ±SD represents the number of biological replicates n = 58 in the control group, n = 11 in the early stage, and n = 41 in the late stage. This indicates a comparison with the normal group (Control). <0.05, <0.01 and <0.001; # This indicates a comparison with the early stage. # P<0.05.

[0025] Figure 5 For plasma fluorescence lifetime detection; among which, Figure 5 A represents the fluorescence decay curve of porphyrin in the plasma of neuroblastoma patients; Figure 5 B is the fluorescence decay curve of plasma flavin / lipofuscin (the excitation and emission spectra of plasma flavin / lipofuscin basically overlap).

[0026] Figure 6 Quantification of fluorescence lifetime components: among which, Figure 6 A-6F represent the porphyrin fluorescence lifetime components excited at 400 nm. And the quantitative determination of the corresponding component proportions a1, a2, and a3; Figure 6 G-6L are the fluorescence lifetime components of flavin / lipofuscin excited at 450 nm. And the quantitative determination of the corresponding component proportions a1, a2, a3; experimental data are presented in the form of... ±SD represents the number of biological replicates n = 58 in the control group, n = 11 in the early stage, and n = 41 in the late stage. This indicates a comparison with the normal group (Control). <0.05, <0.01 and <0.001; # This indicates a comparison with the early stage. # P<0.05 and ### P<0.001.

[0027] Figure 7 Based on plasma tryptophan fluorescence intensity and flavin / lipofuscin fluorescence lifetime ( The ROC curves plotted from the predicted values ​​were used for the differential diagnosis of neuroblastoma at different stages (normal control and early-to-late stage neuroblastoma). Detailed Implementation

[0028] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0030] In the following examples, all clinical plasma specimens were obtained from pediatric neuroblastoma patients, and their collection was approved by the Institutional Review Committee of the Affiliated Women and Children's Medical Center of Guangzhou Medical University (No.: Sui Fu Er Ke Lun Tong Zi

[2025] No. 348B00).

[0031] Terminology Explanation: (1) Neuroblastoma: a malignant solid tumor of the extracranial region of children that originates from primitive neural crest cells of the neuroectoderm and is highly heterogeneous.

[0032] (2) Label-free fluorescence technology: a technology that directly detects the spontaneous fluorescence signal of endogenous fluorophores (such as amino acids, coenzymes, porphyrins, etc.) in a sample without relying on exogenous fluorescent probes.

[0033] (3) Autofluorescence: Fluorescent signals emitted naturally by certain endogenous molecules in biological samples when excited by light of a specific wavelength.

[0034] (4) Endogenous fluorescent groups: Molecules that are naturally occurring in organisms and have fluorescent properties, such as tryptophan, tyrosine, phenylalanine, porphyrin, flavin, lipofuscin, etc.

[0035] (5) Fluorescence spectrum: A spectrum describing the fluorescence intensity of a substance at different excitation / emission wavelengths.

[0036] (6) Fluorescence lifetime: The average time a fluorescent molecule remains in the excited state before returning to the ground state, usually measured in nanoseconds (ns).

[0037] (7) Tryptophan (TRP): an aromatic amino acid that is one of the main contributors to autofluorescence in plasma.

[0038] (8) Tyrosine (TYR): An aromatic amino acid with certain endogenous fluorescence properties.

[0039] (9) Phenylalanine (PHE): An aromatic amino acid with weak endogenous fluorescence.

[0040] (10) Porphyrin: a prosthetic group in heme proteins such as hemoglobin, which has endogenous fluorescence.

[0041] (11) Flavin: Coenzymes (such as FAD and FMN) that participate in redox reactions and have endogenous fluorescence.

[0042] (12) Lipofuscin: an senile pigment-like substance associated with lipid peroxidation, which has endogenous fluorescence.

[0043] (13) ROC curve: Receiver operating characteristic curve, used to assess the discriminative ability of diagnostic indicators.

[0044] (14) AUC: Area under the ROC curve, which reflects the diagnostic accuracy. The closer it is to 1, the better the performance.

[0045] The present invention achieves its objective through the following technical solutions: Plasma is the preferred material for non-invasive diagnostics, not only because it is convenient and can be repeatedly extracted during disease monitoring and treatment, but more importantly, because human plasma contains various endogenous fluorophores, including enzymes, lipids, and porphyrins. The expression levels of these endogenous fluorophores can reflect metabolic changes caused by physiological abnormalities, giving plasma autofluorescence detection the advantages of being label-free, rapid, and low-cost. This technology holds promise for a major breakthrough in the early diagnosis and treatment monitoring of tumors.

[0046] Common endogenous fluorophores in blood plasma include aromatic amino acids, porphyrins in hemoglobin, coenzymes involved in metabolic and functional processes, lipid pigments, and nutritional or symbiotic vitamins. These substances can produce autofluorescence under specific wavelengths of light, and their levels are closely related to disease states.

[0047] Numerous studies have demonstrated the potential of blood autofluorescence to distinguish between normal and diseased tissues. To screen for more biomarkers suitable for the label-free early diagnosis of neuroblastoma, this invention performed autofluorescence detection on plasma from a large number of cancer patients. An excitation wavelength scanning method was employed, scanning from long wavelengths (which excite fewer molecular types) to short wavelengths, covering a range of 561 nm to 280 nm. After initial screening, the following specific excitation wavelengths were determined for measurement: 561, 520, 490, 460, 410, 390, 370, 330, 300, and 280 nm. Fluorescence spectroscopy results showed that the difference in autofluorescence intensity was most significant under excitation at 280 nm. Compared with the normal group, the autofluorescence intensity of plasma from patients with early and late-stage cancers was significantly reduced under excitation at 280 nm, with the weakest signal observed in early-stage patients. However, no significant difference in autofluorescence intensity was observed under excitation at 300 nm, 490 nm, and 561 nm. Under excitation conditions of 330 nm, 370 nm, 390 nm, 410 nm, 460 nm and 520 nm, the autofluorescence intensity of early tumor plasma was not different from that of the normal group; only the autofluorescence intensity of late tumor plasma was significantly higher than that of the normal group or the early group.

[0048] Aromatic amino acids are primarily excited at 280 nm, among which tryptophan (TRP), tyrosine (TYR), and phenylalanine (PHE) possess endogenous fluorescence properties. To determine which component is the major contributor to differential metabolic fluorescence, further plasma amino acid-targeted metabolomics analysis was performed. The results showed that among TRP, TYR, and PHE, TRP exhibited the most significant changes. Compared to the normal control group, plasma TRP levels were significantly lower in both early and late-stage tumor patients, with the lowest levels observed in early-stage patients, significantly lower than in late-stage patients. This trend perfectly aligns with the decrease in 280 nm autofluorescence intensity, indicating that TRP is the primary source of differential metabolic fluorescence. Although plasma TYR levels in early and late-stage tumor patients were also significantly lower than in the normal control group, the decrease was less pronounced than that of TRP, and there was no difference between early and late-stage patients; while there was no significant difference in PHE levels among the three groups (normal, early, and late). These results suggest that TYR contributes only partially to differential metabolic fluorescence, while PHE contributes virtually nothing.

[0049] To further verify the metabolic differences between plasma samples from normal controls and patients with early and late-stage neuroblastoma, fluorescence lifetime detection was performed. Due to instrument limitations (only 400 nm and 450 nm lasers were available), the 400 nm laser primarily excited porphyrins, while the 450 nm laser could simultaneously excite flavin and lipofuscin. Therefore, this study adopted the following strategy: using 400 nm excitation light, the fluorescence lifetime signal of porphyrins was detected in the 625-675 nm channel; using 450 nm excitation light, the fluorescence lifetime signals of flavin and lipofuscin were simultaneously collected in the 525-575 nm channel. The results showed that under 400 nm excitation, there was no significant difference in the fluorescence lifetime of porphyrins among the groups; however, under 450 nm excitation, the plasma fluorescence lifetime of late-stage patients was significantly longer than that of the normal control group and early-stage patients. Further fluorescence lifetime component analysis showed that, compared with normal controls, the three fluorescence lifetime components of porphyrins in the plasma of early and late-stage tumor patients (…) The values ​​of the three fluorescence lifetime components (a1, a2, a3) and their relative proportions did not show significant changes. However, in late-stage patients, the values ​​of the three fluorescence lifetime components jointly contributed by flavin and lipofuscin were significantly higher than those in early-stage patients and the normal control group, although there were no statistically significant differences in the proportions (a1, a2, a3) of each component among the groups. The specific contributions of flavin and lipofuscin to the aforementioned differential fluorescence lifetime components need to be evaluated separately with a larger sample size.

[0050] Finally, the significant optical parameters selected were TRP fluorescence intensity and flavin / lipofuscin fluorescence lifetime. The combined optical parameters were used to perform binary logistic regression analysis to calculate the predicted probability, and ROC curves were used to evaluate its predictive value in the diagnosis of early neuroblastoma. The results showed that the combined optical parameters had an accuracy (AUC) of 0.77 in distinguishing normal controls from early neuroblastoma; an accuracy (AUC) of 0.92 in distinguishing normal controls from late neuroblastoma; and an accuracy (AUC) of 0.99 in distinguishing early and late neuroblastoma. The relatively low AUC (0.77) in distinguishing early neuroblastoma from normal controls may be related to the smaller clinical sample size of early-stage patients. In summary, the TRP fluorescence intensity combined with flavin / lipofuscin fluorescence lifetime (…)… It has shown potential in the diagnosis of neuroblastoma, and may play a key role, especially in the early diagnosis in the future.

[0051] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0052] Example 1: Measurement and Analysis of Blood Plasma Autofluorescence Spectroscopy 1. Experimental Method: (1) The plasma fluorescence spectrum was measured using an FL970 fluorescence spectrophotometer, with both excitation and emission bandwidths set to 2.5 nm. An excitation wavelength scanning method was employed, scanning from longer wavelengths (for fewer excited molecules) to shorter wavelengths. Specific excitation wavelengths measured were: 561, 520, 490, 460, 410, 390, 370, 330, 300, and 280 nm. Sample preparation: 160 µL of plasma was diluted with 480 µL of PBS, mixed thoroughly, and then 600 µL was injected into a quartz cuvette for measurement. The PBS spectrum at the corresponding wavelength was measured simultaneously for each measurement, and the plasma fluorescence intensity was subtracted from the PBS background value to correct for batch variations.

[0053] (2) Observation index: plasma fluorescence spectrum intensity.

[0054] 2. Experimental Results Fluorescence spectroscopy results showed that the difference in autofluorescence intensity was most significant under excitation at 280 nm. Figure 1 AJ). Compared with the normal group, the autofluorescence intensity of plasma from patients with early and late-stage tumors was significantly reduced under excitation at 280 nm, with the weakest signal observed in early-stage patients. However, there was no significant difference in autofluorescence intensity under excitation at 300 nm, 490 nm, and 561 nm. Under excitation conditions of 330 nm, 370 nm, 390 nm, 410 nm, 460 nm, and 520 nm, the autofluorescence intensity of plasma from early-stage tumors was not different from that of the normal group; only the autofluorescence intensity of plasma from late-stage tumors was significantly higher than that of the normal group or the early-stage group (AJ). Figure 1 K).

[0055] Example 2: Plasma Amino Acid Targeted Metabolite Profiling 1. Experimental Method: 1.1 Sample Preparation (1) Take a plasma sample, thaw it at room temperature, and vortex it for 10 seconds to mix.

[0056] (2) Take 10 μL of plasma sample, place it in a 1.5 mL centrifuge tube, add 10 μL of ultrapure water, 5 μL of internal standard and 40 μL of isopropanol (containing 0.1% formic acid), and vortex for 2 min.

[0057] (3) Place the centrifuge tube in a low-temperature centrifuge and centrifuge at 4 ℃ and 12000 rpm for 10 min.

[0058] (4) Take 10 μL of the supernatant, place it in a 1.5 mL centrifuge tube, add 70 μL of borate buffer, and vortex for 1 min.

[0059] (5) Add 20 μL of AccQ Tag derivatizing reagent (Kairos Amino Acid Kit, USA) and shake immediately for 10 s. After 1 min, the excess derivatizing agent hydrolyzes, and the derivatization reaction ends; place the centrifuge tube at 55 ℃ and heat for 10 min; then add 400 μL of ultrapure water and vortex for 1 min to dilute.

[0060] (6) Centrifuge at 4 ℃ and 12000 rpm for 5 min. Transfer 150 μL of the supernatant after centrifugation to a liquid chromatography vial for analysis.

[0061] 1.2 Preparation of Standards (1) Using the gradient dilution method, amino acid solutions with concentrations of 400, 200, 100, 40, 20, 10, 4, 2 and 1 μmol / L were prepared sequentially.

[0062] (2) Take 20 μL of sample, 5 μL of internal standard and 40 μL of isopropanol (0.1% formic acid), and vortex for 2 min.

[0063] (3) Place the centrifuge tube in a low-temperature centrifuge and centrifuge at 4 ℃ and 12000 rpm for 10 min.

[0064] (4) Take 10 μL of the supernatant and place it in a 1.5 mL centrifuge tube. Add 70 μL of borate buffer and 20 μL of LaccQ Tag derivatizing reagent (Kairos Amino Acid Kit). Shake immediately for 10 s. After 1 min, the excess derivatizing agent hydrolyzes, and the derivatization reaction ends. Place the centrifuge tube at 55 °C for 10 min. Then, add 400 μL of water to dilute.

[0065] (5) Take 100 μL of the supernatant and transfer it to a liquid chromatography vial for testing.

[0066] 1.3 Chromatographic conditions Chromatographic separation was performed using Waters ACQUITY UPLC I-CLASS ultra-high performance liquid chromatography under the following conditions: Column: Waters UPLC HSS T3 (1.8 μm, 2.1 mm × 150 mm); Mobile phase: Phase A (water, 0.1% formic acid), Phase B (acetonitrile); Elution gradient: See Table 1; Flow rate: 0.5 mL / min; Injection volume: 5.0 µL; Column temperature: 50 ℃.

[0067] Table 1: Elution Gradient Table 1.4 Mass Spectrometry Conditions Mass spectrometry analysis was performed using a Waters XEVO TQ-S Micro tandem quadrupole mass spectrometer system. The positive ion source voltage was 1.5 kV and the cone voltage was 20 V; the desolventizing temperature was 600 °C and the desolventizing gas flow rate was 1000 L / h; the cone gas flow rate was 10 L / h.

[0068] 1.5 Targeted Data Processing The peak area was calculated using MassLynx quantitative software, with a retention time tolerance of 15 s. The quantitative results were obtained using the standard curve method.

[0069] 1.6 Observation indicators: Expression level of amino acid-targeting metabolites.

[0070] 2. Experimental Results The results showed that among the three amino acids tryptophan (TRP), tyrosine (TYR), and phenylalanine (PHE), TRP showed the most significant change. Figures 2-4 Compared with the normal control group, the plasma TRP levels in patients with early and late-stage tumors were significantly reduced, with the lowest levels observed in early-stage patients, significantly lower than in late-stage patients. This trend is entirely consistent with the decrease in 280 nm autofluorescence intensity, indicating that TRP is the main source of differential metabolic fluorescence. Although the plasma TYR levels in patients with early and late-stage tumors were also significantly lower than in the normal control group, the decrease was less than that of TRP, and there was no difference between early and late-stage patients; while there was no significant difference in PHE levels among the three groups (normal, early, and late). These results suggest that TYR contributes only partially to differential metabolic fluorescence, while PHE contributes virtually nothing.

[0071] Example 3: Measurement of plasma fluorescence lifetime 1. Experimental Method: (1) The emission spectra of fluorescent molecules often overlap, making it difficult to accurately distinguish and qualitatively identify the components; however, fluorescence lifetime detection can achieve specific identification of fluorescent substances. In addition, when fluorescent molecules couple or bind with proteins, their fluorescence lifetime will change characteristically, so fluorescence lifetime analysis can be used to identify coupled fluorescent metabolites. In this experiment, a Mini-Tau fluorescence lifetime spectrometer (Edinburgh Instruments) equipped with a TCSPC module was used. The detection time range was set to 50 ns and the number of channels was 1024. Acquisition was stopped when the peak count reached 7000. Then, fluorescence lifetime detection was performed sequentially under the following excitation wavelength and detection channel conditions: (1) 450 nm excitation, 525-575 nm channel for emission signal collection; (2) 400 nm excitation, 625-675 nm channel for emission signal collection; the laser intensity was adjusted to a suitable level by an optical attenuator during the detection process. A triple exponential decay model was used. Fit the decay curve to obtain the characteristic lifetime ( The decay curves and fitting data were derived by taking the values ​​of the corresponding fluorescence lifetime components (a1, a2, a3) and their respective percentages. Where t represents the decay time. Fluorescence intensity at a certain moment; : The decay rate (fluorescence lifetime) of three different fluorescent molecules (porphyrin, flavin, lipofuscin); the proportion of each lifetime component a1, a2, a3; e: natural constant (Euler number).

[0072] (2) Observation indicators: fluorescence lifetime decay curves of plasma porphyrin and flavonoid / lipofuscin.

[0073] 2. Experimental Results Due to instrument limitations (only 400 nm and 450 nm lasers were available), the 400 nm laser specifically excited porphyrins, while the 450 nm laser simultaneously excited flavins and lipofuscins. Therefore, this study employed the following detection strategy: using 400 nm as the excitation wavelength, fluorescence lifetime signals of porphyrins were acquired in the 625–675 nm detection channel; using 450 nm as the excitation wavelength, fluorescence lifetime signals of flavins and lipofuscins were simultaneously acquired in the 525–575 nm detection channel. The results showed that under 400 nm excitation, there was no significant difference in the fluorescence lifetime of porphyrins among the groups; however, under 450 nm excitation, the plasma fluorescence lifetime of late-stage patients was significantly longer than that of the normal control group and early-stage patients. Figure 5 AB).

[0074] Example 4: Analysis of plasma fluorescence lifetime components 1. Experimental Method: (1) The data obtained by the Mini-Tau fluorescence lifetime spectrometer in Example 3 were analyzed using the triple exponential decay model. Fit the decay curve to obtain the characteristic lifetime ( The decay curves and fitting data were derived from the corresponding percentages of fluorescence lifetime components (a1, a2, a3).

[0075] (2) Observation indicators: fluorescence lifetime components of plasma porphyrin and flavonoid / lipofuscin.

[0076] 2. Experimental Results Further fluorescence lifetime component analysis showed that, compared with normal controls, the three fluorescence lifetime components of porphyrin in the plasma of patients with early and late-stage tumors ( The proportions of a1, a2, and a3 did not show significant changes. Figure 6AF). However, in advanced patients, the values ​​of the three fluorescence lifetime components jointly contributed by flavin and lipofuscin were significantly higher than those in early-stage patients and normal controls, although there were no statistically significant differences in the proportions (a1, a2, a3) of each component among the groups. Figure 6 The respective contributions of flavonoids and lipofuscin to the aforementioned differential lifespan components require further analysis and evaluation with a larger sample size.

[0077] Example 5: Establishment of an early diagnostic model for neuroblastoma 1. Experimental Method: (1) The binary classification accuracy of various optical parameters, including fluorescence lifetime fitting parameters, was calculated using logistic regression and leave-one-out cross-validation in SPSS software. a1, a2, a3, and fluorescence intensity were used. Disease prediction probabilities were calculated using a binomial logistic regression model in SPSS software. For each individual or combined indicator, receiver operating characteristic (ROC) curves were plotted using GraphPad Prism 10.0 software, and the area under the curve (AUC) was calculated to evaluate their performance in distinguishing between healthy, early- and late-stage tumors. The optimal diagnostic threshold, sensitivity, and specificity for each indicator were determined using the ROC curves.

[0078] (2) Observation indicators: ROC and AUC.

[0079] 2. Experimental Results The significant optical parameters selected were TRP fluorescence intensity and flavin / lipofuscin fluorescence lifetime. The combined optical parameters were used to perform binary logistic regression analysis to calculate the predicted probability, and ROC curves were used to evaluate its predictive value in the diagnosis of early neuroblastoma. The results showed that the accuracy (AUC) of the combined optical parameters in distinguishing between normal controls and early neuroblastoma was 0.77; the accuracy (AUC) in distinguishing between normal controls and late neuroblastoma was 0.92; and the accuracy (AUC) in distinguishing between early and late neuroblastoma was 0.99. The relatively low AUC (0.77) in distinguishing early neuroblastoma from normal controls may be related to the smaller clinical sample size of early-stage patients. Figure 7 In summary, TRP fluorescence intensity combined with flavin / lipofuscin fluorescence lifetime ( It has shown potential in the diagnosis of neuroblastoma, and may play a key role, especially in the early diagnosis in the future.

[0080] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The application of a combination of optical detection parameters in the preparation of diagnostic equipment or procedures for pediatric neuroblastoma, characterized in that, The optical detection parameter combination is: the fluorescence intensity of tryptophan in body fluid detected by a 280 nm excitation wavelength, and the fluorescence lifetime fitting component of flavin / lipofuscin in body fluid detected by a 450 nm excitation wavelength. .

2. The application according to claim 1, characterized in that, The body fluids include plasma, serum, urine, or cerebrospinal fluid.

3. The application according to claim 1, characterized in that, The aforementioned diagnostic equipment or procedure for pediatric neuroblastoma can be used for the early diagnosis of neuroblastoma or to differentiate between early and late-stage neuroblastoma. Specifically, compared to the general population, the autofluorescence intensity of body fluids from patients with both early and late-stage tumors is significantly reduced under 280 nm excitation, with the signal being relatively weaker in early-stage patients. The fitted component of the flavin / lipofuscin fluorescence lifetime in the body fluids of patients with late-stage tumors... The rate was significantly higher than that of patients with early-stage cancer.

4. The application according to claim 1, characterized in that, The diagnostic testing equipment includes a fluorescence spectrophotometer and a fluorescence lifetime spectrometer equipped with a TCSPC module.

5. The application according to claim 4, characterized in that, Fluorescence lifetime fitting components The acquisition method is as follows: using a fluorescence lifetime spectrometer equipped with a TCSPC module, the detection time is set to 50 ns and the number of channels to 1024. Acquisition is stopped when the peak count reaches 7000. Then, the excitation wavelength is set to 450 nm, and the emission signals of flavin / lipofuscin are acquired in the 525~575 nm channel. Finally, a triple exponential decay model is used. It is obtained by fitting the decay curve.

6. The application according to claim 4, characterized in that, The method for obtaining the fluorescence intensity of tryptophan in body fluids is as follows: autofluorescence spectroscopy of body fluid samples is performed using a fluorescence spectrophotometer, with the excitation wavelength set to 280 nm and the excitation and emission bandwidths both set to 2.5 nm, thereby measuring the autofluorescence intensity corresponding to tryptophan in body fluids.

7. The application according to claim 6, characterized in that, The background spectrum of the PBS blank was measured simultaneously during the detection process, and background interference was subtracted.

8. A non-invasive early identification method for neuroblastoma based on plasma optical characteristics, characterized in that, The method does not involve the field of disease diagnosis and treatment, and specifically includes the following steps: S1. The autofluorescence spectrum of plasma samples was detected by a fluorescence spectrophotometer. The excitation wavelength was set to 280 nm, and the autofluorescence intensity corresponding to plasma tryptophan was measured. S2. A fluorescence lifetime spectrometer equipped with a TCSPC module was used to detect the fluorescence lifetime of plasma samples. An excitation wavelength of 450 nm was set, and the emission signals of flavin / lipofuscin were acquired in the 525–575 nm channel. A triple exponential decay model was then used. Fluorescence lifetime parameters of flavin / lipofuscin were obtained by fitting decay curves. ; S3. Measure the fluorescence intensity of tryptophan and the fluorescence lifetime parameters of flavin / lipofuscin under 280 nm excitation. They are used together as input variables to construct a logistic regression diagnostic model; S4. The logistic regression diagnostic model is used to differentiate between normal individuals, early-stage neuroblastoma patients, and late-stage neuroblastoma patients.

9. A non-invasive early diagnostic method for neuroblastoma based on plasma optical characteristics according to claim 8, characterized in that, The criteria for the logistic regression diagnostic model are as follows: the AUC for distinguishing between normal individuals and early-stage neuroblastoma is 0.77; the AUC for distinguishing between normal individuals and late-stage neuroblastoma is 0.92; and the AUC for distinguishing between early-stage and late-stage neuroblastoma is 0.99.

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