Upconversion fluorescent probe, isoniazid detection method and portable isoniazid detection sensor

Abstract

The application discloses an up-conversion fluorescent probe, an isoniazid detection method and a portable isoniazid detection sensor and belongs to the field of biological detection. The up-conversion fluorescent probe is composed of lanthanide-doped core-shell structure up-conversion nanoparticles subjected to acid pickling treatment and p-dimethylaminobenzaldehyde, wherein the core particle diameter of the up-conversion nanoparticles is 25.85-28.85 nm, and the shell layer thickness is 0.98-1.38 nm. The lanthanide-doped core-shell structure up-conversion nanoparticles are used as an energy donor, and the p-dimethylaminobenzaldehyde is used as a specific recognition molecule, and ratio-type fluorescence detection is realized through an inner filter effect. Isoniazid reacts with DMAAB to generate a Schiff base, which causes fluorescence quenching at 475 nm and no change in 655 nm fluorescence, so that the fluorescence color changes from blue to red. Due to the near-infrared excitation characteristics of the up-conversion nanoparticles, the up-conversion nanoparticles are not easily disturbed by background fluorescence, so that rapid quantitative detection of isoniazid in whole blood is realized, and the detection limit is as low as 2.4 μm. The application is simple in operation, strong in anti-interference and suitable for clinical individualized drug monitoring.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically an upconversion fluorescent probe, a method for detecting isoniazid, and a portable isoniazid detection sensor. Background Technology

[0002] Isoniazid, as a first-line anti-tuberculosis drug, is widely used globally due to its high selectivity and significant efficacy against Mycobacterium tuberculosis. In clinical practice, isoniazid not only effectively controls tuberculosis symptoms but also exhibits high safety during treatment due to its low toxicity. However, long-term use of isoniazid may lead to drug accumulation in the body, a process directly associated with chemical liver injury and peripheral neuropathy. Furthermore, isoniazid has a narrow therapeutic window (effective therapeutic concentration of 3-6 μg / mL, toxicity threshold >10 μg / mL), and N-acetyltransferase 2 (NAT2) gene polymorphism leads to differences in fast / slow acetylation phenotypes among individuals, resulting in blood drug concentration fluctuations of 5-20 times at the same dose. This metabolic heterogeneity directly leads to treatment failure or adverse drug reactions in some patients. Although the efficacy of isoniazid far surpasses other alternative drugs, the contradiction between its toxicity risks and the need for individualized medication highlights the urgency of real-time blood drug concentration monitoring. Current detection techniques such as HPLC and UV spectrophotometry suffer from drawbacks including expensive equipment, complex sample preparation, and the inability to perform real-time monitoring. While upconversion nanoprobes have been used in biological detection, previous probes have only been applicable to serum. The detection method proposed in this invention is based on the chemical reaction between upconversion nanoprobes and isoniazid, which can effectively avoid interference from background fluorescence in whole blood and can be used for the detection of drug concentrations in complex biological systems. Summary of the Invention

[0003] In view of this, the present invention provides an upconversion fluorescent probe, an isoniazid detection method, and a portable isoniazid detection sensor to solve the problems mentioned in the background art, based on upconversion nanoparticles and a fluorescent probe of p-dimethylaminobenzaldehyde for highly sensitive detection of isoniazid concentration in whole blood.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] In a first aspect, the present invention discloses an upconversion fluorescent probe, which is composed of acid-washed lanthanide-doped core-shell structured upconversion nanoparticles and p-dimethylaminobenzaldehyde, wherein the core particle size of the upconversion nanoparticles is 25.85-28.85 nm and the shell thickness is 0.98-1.38 nm.

[0006] As a further aspect of the present invention, the acid washing process specifically involves: subjecting the upconversion nanoparticles to ultrasonic acid washing with concentrated acid, causing their zeta potential value to change from negative to positive, and the potential change amplitude being >50 mV.

[0007] As a further aspect of the present invention, the fluorescence signal fluctuation of the probe is less than 3% within a temperature range of 10-80℃.

[0008] Secondly, this invention discloses a method for detecting isoniazid, comprising the following steps:

[0009] S1. Mix the above upconversion fluorescent probe with concentrated acid to obtain a probe solution;

[0010] S2. Add the whole blood sample to be tested to the probe solution and react for at least 70 seconds to obtain the reaction solution;

[0011] S3. Detect the fluorescence intensity ratio I between 475 nm and 655 nm of the reaction solution. 475 / I 655 ;

[0012] S4. The obtained fluorescence intensity is compared with I 475 / I 655 Substitute the values ​​into the standard curve to calculate the concentration of isoniazid.

[0013] As a further aspect of the present invention: in step S3, the calculation formula for the standard curve is I 475 / I 655 = -0.0089C + 0.98; where C is the isoniazid concentration in μm; I 475 Fluorescence intensity at 475 nm; I 655 The fluorescence intensity is at 655 nm.

[0014] As a further aspect of the present invention, its detection limit is 2.4 μm.

[0015] Thirdly, the present invention discloses a portable isoniazid detection sensor, comprising an immunochromatographic test strip having the above-mentioned upconversion fluorescent probe.

[0016] Furthermore, the portable isoniazid detection sensor also includes:

[0017] An optical excitation and signal acquisition module is used to excite upconversion nanoparticles and separate target fluorescence;

[0018] The cavity is used to house the optical excitation and signal acquisition module;

[0019] The image acquisition module and RGB fluorescence ratio analysis system are used to convert fluorescence color changes into B / R values ​​(blue-red fluorescence intensity ratio) and calculate concentration.

[0020] The optical excitation and signal acquisition module includes a 980 nm laser, an optical filter, and a cuvette holder.

[0021] As a further aspect of the present invention:

[0022] The RGB fluorescence ratio analysis system establishes a linear equation between the B / R value and the isoniazid concentration. The portable isoniazid detection sensor has a detection range of 0-100 μm. The whole blood sample spike recovery rate is 98-110%, and the relative standard deviation is ≤3.2%.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. This invention uses acid-washed hydrophilic core-shell UCNPs (NaYF4:Yb,Tm@NaYF4) as the energy donor and p-dimethylaminobenzaldehyde (DMAAB) as the recognition molecule. Isoniazid reacts with DMAAB under acidic conditions to form a Schiff base, whose 475 nm absorption peak overlaps with the emission spectrum of upconversion nanoparticles (UCNPs), triggering an internal filtration effect (IFE) that leads to selective quenching of fluorescence at 475 nm while the fluorescence at 655 nm remains unchanged, causing the fluorescence color to change from blue to red, thus achieving dual-wavelength ratio detection.

[0025] 2. The probe prepared in this invention can be used to detect the concentration of isoniazid in whole blood. Due to the near-infrared excitation characteristics of upconversion nanoparticles, they are not easily affected by background fluorescence, thus enabling rapid quantitative detection of isoniazid in whole blood, with a detection limit as low as 2.4 μm. This invention is simple to operate, has strong anti-interference capabilities, and is suitable for clinical personalized medication monitoring.

[0026] 4. It features a rapid 70-second response time, a whole blood spike recovery rate of 98-110%, an RSD of ≤3.2%, and a wide detection temperature range (10-80℃) and excellent pH stability.

[0027] 5. This invention combines upconversion nanoprobes with immunochromatographic test strips to construct a portable detection platform. Attached Figure Description

[0028] Figure 1 TEM images and particle size characterization of the upconversion nanoparticles prepared in Example 1;

[0029] Figure 2 XRD, XPS, and zeta potential spectra of the upconversion nanoparticles prepared in Example 1;

[0030] Figure 3 The FTIR spectrum of the material prepared in Example 1 of this invention;

[0031] Figure 4 This is a performance test of the probe sensitivity, selectivity, and anti-interference capabilities in Embodiment 3 of the present invention;

[0032] Figure 5 Stability test of the upconversion probe prepared in Example 3 of the present invention in the temperature range of 10-80℃;

[0033] Figure 6 The stability test of the upconversion probe prepared in Example 3 of the present invention within 140 minutes;

[0034] Figure 7 To determine the reaction efficiency of the upconversion probe prepared in Example 3 of this invention with isoniazid at different pH values, acidic conditions (pH value of 1) were determined to be the optimal reaction environment.

[0035] Figure 8 The response time curve of the probe prepared in Example 3 of the present invention shows that the signal reaches the plateau phase after 70 seconds; where the horizontal axis is time and the vertical axis is the absorbance value of the probe.

[0036] Figure 9 This is a schematic diagram of the probe prepared in Example 4 of the present invention detecting the concentration of isoniazid in whole blood;

[0037] Figure 10 The effect of the probe prepared in Example 4 of this invention in whole blood testing: Figure 10 A and Figure 10 B shows the relationship between the change in fluorescence color gradient and concentration. Figure 10 C shows a linear correlation between the B / R value and concentration. Figure 10 D shows a spiked recovery rate of 101.59-109.5%;

[0038] Figure 11 This is a schematic diagram of the portable isoniazid detection sensor structure of the present invention, wherein... Figure 11 A represents a specific component of the sensor device. Figure 11 B represents the overall structure of the sensor device. Detailed Implementation

[0039] To facilitate understanding of the present invention, a more comprehensive description will be given below with reference to specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0041] Example 1: Preparation of core-shell structured UCNPs

[0042] 1. Nucleosynthesis: ErCl3·6H2O, YbCl3·6H2O, and TmCl3·6H2O were dissolved in an excess of oleic acid / octadecene mixture at a molar ratio of 78:20:2. After deoxygenation at 150°C, 10 mL of NaOH / NH4F methanol solution was added, and the mixture was reacted at 300°C for 1 hour to obtain upconversion nanoparticles (UCNPs).

[0043] 2. Shell coating: The core solution and YCl3 precursor were mixed at a ratio of 1:0.2 and coated with an inert shell of NaYF4 using the same method.

[0044] 3. Acid washing treatment: Sonicate with hydrochloric acid at pH=1 for 1 hour, then centrifuge to disperse the water ( Figure 2 B verified the zeta potential transition.

[0045] Example 2: Preparation of upconversion fluorescent probe

[0046] 50 μL of acid-washed UCNPs were added to a 5 mL centrifuge tube, followed by 10 μL of hydrochloric acid (pH = 1) and 1 mL of 0.1 mol / L dimethylaminobenzaldehyde solution to prepare the probe solution. Ethanol was added to the probe solution to adjust the final volume to 2 mL. Equal volumes of isoniazid solutions of different concentrations were then introduced into the probe solution and thoroughly mixed. After the reaction, changes in absorbance and fluorescence intensity were recorded. Each experiment was performed five times to ensure reproducibility. Furthermore, the corresponding color gradient changes at different isoniazid concentrations were visually observed under ambient light and darkness conditions.

[0047] Example 3: Probe Performance Testing

[0048] The pH of the probe system was adjusted using hydrochloric acid and sodium hydroxide, and the degree of reaction between the probe and isoniazid was evaluated under different pH conditions to determine the optimal pH value. The results are shown in [Figure number missing]. Figure 7 Furthermore, the thermal stability of the probe was evaluated within a temperature range of 10–80 °C, and the effect of temperature on its performance was investigated. The results are shown in [Figure number missing]. Figure 5 .

[0049] First, prepare isoniazid standard solutions (water as solvent). At room temperature, mix 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μm isoniazid standard solutions with 2 mL of probe solution, record the fluorescence spectra, and linearly fit the isoniazid concentration C with I0. 475 / I 655 The standard curve I is obtained. 475 / I 655 = -0.0089C + 0.98.

[0050] The probe performance test results are as follows:

[0051] 1. Sensitivity: I 475 / I 655 Linear correlation with 0-100 μm isoniazid (e.g.) Figure 4 (as shown in C).

[0052] 2. Specificity: Signal change <5% under 100 μm interfering substances (ascorbic acid / dopamine, etc.) (e.g.) Figure 4 (as shown in D).

[0053] 3. Stability: Signal fluctuation <3% within 10-80℃ and 140 minutes (e.g., Figure 5 and Figure 6 (As shown).

[0054] 4. Response time < 70 seconds, supports real-time monitoring.

[0055] Example 4: Detection of Isoniazid Spiked in Whole Blood

[0056] 1. Take whole blood from healthy individuals, spike it with isoniazid (concentration gradient range of 10-70 μm), add 2 ml of the probe solution obtained in Example 2, and react for 70 seconds.

[0057] 2. The B / R value was measured using a smartphone platform, and the recovery rate was calculated to be 101.59-109.5% (e.g., Figure 10 (as shown in D).

[0058] 3. Compare the isoniazid concentration results obtained above with those obtained by HPLC detection. The relative error between the two is <5% (e.g., Figure 10 (as shown in C).

[0059] Example 5: Portable Isoniazid Detection Sensor

[0060] like Figure 11 As shown, this embodiment discloses a portable isoniazid detection sensor. Please refer to [link / reference]. Figure 11 A. This portable isoniazid detection sensor includes the following components:

[0061] Immunochromatographic test strips with upconversion fluorescent probes (i.e. Figure 11 (The immunochromatographic apparatus in A); the porous substrate of the test strip is a nitrocellulose membrane. The probe solution is sprayed onto the test strip by an inkjet printer and then air-dried.

[0062] Optical excitation and signal acquisition module (i.e. Figure 11 The 980nm laser in A is used to excite upconversion nanoparticles and separate the target fluorescence;

[0063] A cavity is used to form a dark chamber and to house the optical excitation and signal acquisition module and the test strip;

[0064] Image acquisition module and RGB fluorescence ratio analysis system (built into) Figure 11 In a smartphone (as described in A), the system converts the acquired fluorescence color changes into B / R values ​​and calculates the concentration. This image acquisition module and RGB fluorescence ratio analysis system can be implemented via a smartphone or tablet, capturing the original image through a camera. An optical filter is physically mounted in front of the camera. The optical excitation and signal acquisition module includes a 980 nm laser, an optical filter, and a cuvette holder.

[0065] Please see Figure 11 B shows the overall structure of the assembled portable isoniazid detection sensor.

[0066] During the test, the sample is dropped onto the test strip, and then the test strip is inserted into the cavity and closed to form a completely dark detection environment. After 70 seconds, the camera is turned on to photograph the test strip in the dark chamber. The fluorescence color change of the acquired image is converted into a B / R value by the RGB fluorescence ratio analysis system, and the concentration of isoniazid in the sample is calculated.

[0067] The materials prepared according to the embodiments of the present invention and their performance tests were characterized, as detailed below:

[0068] Figure 1 TEM images and particle size characterization of the upconversion nanoparticles prepared in Example 1, wherein: Figure 1 A and Figure 1 B shows the transmission electron microscope (TEM) morphology of the core and core-shell upconversion nanoparticles, indicating that the synthesized nanoparticles all have uniform morphological characteristics. Figure 1 C shows the lattice fringes of the core-shell upconversion nanoparticles under a high-resolution transmission electron microscope (HRTEM), confirming the excellent crystal quality of the nanoparticles. Clear lattice fringes can be observed, which are in perfect agreement with the characteristics of standard crystal planes. Figure 1 D is the elemental analysis diagram of the core-shell upconversion nanoparticles. Figure 1 E and Figure 1F represents the particle size distribution of the core and core-shell nanoparticles. The core particle size is 27.35 nm, and the overall particle size of the core-shell structure is 28.53 nm. This indicates the formation of a uniform inert shell with a thickness of approximately 1.18 nm and a narrow particle size distribution, demonstrating good size consistency and controllability.

[0069] Figure 2 The XRD, XPS, and zeta potential spectra of the upconversion nanoparticles prepared in Example 1 are shown below: Figure 2 Figure A shows the X-ray diffraction analysis of the core and core-shell upconversion nanoparticles and the corresponding standard card. By comparing the XRD diffraction peaks of the particle core and core-shell structure with the standard card (16-0334), it was confirmed that the synthesized particles have a standard hexagonal phase structure. Figure 2 B shows the zeta potential analysis of the core-shell nanoparticles before and after acid washing. The zeta potential of the particles before acid washing was -15.38 mV, while the zeta potential after acid washing jumped to 37.6 mV, indicating that the particle surface changed from negative charge to positive charge, which confirms the removal of oleic acid ligands. Figure 2 C- Figure 2 I is the XPS analysis diagram of the upconversion nanoparticles. The strong absorption peaks at 1073.82 eV, 160.99 eV, 686.56 eV, 187.54 eV and 286.28 eV correspond to the binding energies of Na 1s, Y 3d, F 1s, Yb 4d and C 1s, respectively, further confirming the chemical composition and structure of the particles.

[0070] Figure 3 This is the FTIR spectrum of the material prepared in Example 1 of the present invention. The blue line corresponds to the infrared spectrum of the particles before acid washing, at 2927 cm⁻¹. -1 and 2855 cm -1 The absorption peaks are attributed to the stretching vibrations of the methylene group in the oleic acid ligand, and the magenta lines correspond to the infrared spectra of the particles after acid washing. The significant disappearance of these characteristic absorption peaks indicates that the oleic acid ligand has been successfully removed.

[0071] Figure 4 This is a performance test of the probe sensitivity, selectivity, and anti-interference in Embodiment 3 of the present invention. Figure 4 A and Figure 4 B represents the gradient changes in fluorescence intensity and absorbance of the upconversion nanoprobe with the addition of isoniazid. Under fixed probe concentration conditions, with increasing isoniazid concentration gradient (0-100 μm), the upconversion fluorescence intensity at 475 nm exhibits concentration-dependent quenching, while the fluorescence at 655 nm remains stable. This specific response forms a unique ratiometric signal (I0). 475 / I 655 ). Figure 4C represents the ratio of probe fluorescence intensity and the linear relationship between absorbance and isoniazid concentration; both modes exhibit good linearity. Figure 4 D represents the selectivity and anti-interference value of the probe. Even when multiple interfering substances coexist, the probe can still efficiently detect isoniazid without being significantly interfered with by other components.

[0072] Figure 5 The stability of the upconversion probe prepared in Example 3 of this invention at different temperatures. Wherein: Figure 5 A is the curve showing the relationship between the fluorescence intensity of the upconversion fluorescent probe and its wavelength. Figure 5 B is the fluorescence intensity curve of the upconversion fluorescent probe as a function of temperature. Figure 5 C is the curve showing the relationship between the UV absorbance of the upconversion fluorescent probe and wavelength. Figure 5 D is the curve showing the change in UV absorbance of the upconversion fluorescent probe as a function of temperature. When the temperature gradually increases from 10℃ to 80℃, the fluorescence intensity and UV absorbance of the probe show little change, with no significant fluctuations or attenuation, indicating that the probe has good stability under different temperature conditions. This result demonstrates that the probe can maintain its original performance over a relatively wide temperature range.

[0073] Figure 6 The stability of the upconversion probe prepared in Example 3 of this invention at different times; wherein: Figure 6 A is the curve showing the relationship between the fluorescence intensity of the upconversion fluorescent probe and its wavelength. Figure 6 B is the fluorescence intensity curve of the upconversion fluorescent probe over time. Figure 6 C is the curve showing the relationship between the UV absorbance of the upconversion fluorescent probe and wavelength. Figure 6 D is the curve showing the change in UV absorbance of the upconversion fluorescent probe over time. No significant changes were observed in fluorescence intensity and UV absorbance between 0 and 140 minutes, indicating that the probe maintains relatively stable performance over a longer period. This result further verifies the long-term stability of the probe, ensuring reliable detection results even when exposed to prolonged reaction conditions in actual detection processes.

[0074] Figure 7 The stability of the upconversion probe prepared in Example 3 of this invention at different pH values ​​is shown in the figure. As can be seen from the figure, the probe reacts faster and more efficiently in an acidic environment. The acidic environment effectively promotes the nucleophilic reaction between isoniazid and p-dimethylaminobenzaldehyde, thereby accelerating the Schiff base formation process. Therefore, properly adjusting the pH value of the reaction system is crucial for achieving probe sensitivity and accuracy.

[0075] Figure 8The optimal reaction time for preparing the probe in Example 3 of this invention is shown in the figure. The reaction system reaches a stable state approximately 70 seconds after the reaction of isoniazid and p-dimethylaminobenzaldehyde. This means that the probe can complete the reaction in a short time, and any fluctuations during the reaction process can be quickly stabilized, ensuring the high efficiency and accuracy of the probe in practical applications. This result further demonstrates the rapid response characteristics of the probe in the reaction, which helps to improve detection efficiency.

[0076] Figure 9 This is a schematic diagram illustrating the detection of isoniazid concentration in whole blood using the probe prepared in Example 4 of this invention. The probe solution is first loaded onto an absorbent pad and bonded to a backing plate to construct the detection area. Subsequently, the constructed immunochromatographic device is combined with existing 3D printing technology to develop a portable sensing device (such as...). Figure 11 As shown in the figure, it is specifically designed for rapid detection of isoniazid in complex environments, making on-site detection of isoniazid more efficient and reliable.

[0077] Figure 10 The effect of the probe prepared in Example 4 of the present invention in whole blood testing. Figure 10 A and Figure 10 B shows the correlation between the isoniazid concentration gradient and the probe fluorescence color change after spiking experiments with different concentrations of isoniazid, as well as the division of clinical treatment efficacy and dangerous concentration ranges. Figure 10 C represents the relationship between different concentrations of isoniazid spiking and the B / R ratio. The data shows that the B / R value changes significantly with increasing isoniazid concentration, exhibiting a good linear relationship within a certain range. Furthermore, when the sample detected by the sensor platform has a reddish tint, it indicates that the concentration of isoniazid in whole blood has approached or reached a dangerous level, requiring vigilance. This phenomenon not only verifies the sensitivity and accuracy of the sensor platform but also demonstrates its effectiveness in practical applications. Figure 10 D represents the spiked recovery results of the probe in fluorescence mode. When 10 μm, 30 μm, 50 μm, and 70 μm isoniazid were added, the recoveries of the samples were 109.5%, 100.4%, 100.04%, and 101.59%, respectively, and the relative standard deviations remained between 2.5% and 3.2%, indicating that the platform has high accuracy and reliability in detecting real samples.

[0078] Figure 11 This is a schematic diagram of a portable isoniazid detection sensor. Figure 11 A illustrates the specific components of this sensor structure. Figure 11 B is a schematic diagram of the assembly structure of the portable isoniazid detection sensor before testing.

[0079] Although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0080] Therefore, the above description is only a preferred embodiment of this application and is not intended to limit the scope of this application; that is, all equivalent modifications made in accordance with the scope of the claims of this application shall be within the protection scope of the claims of this application.

Claims

1. An upconversion fluorescent probe, characterized in that, It consists of lanthanide-doped core-shell upconversion nanoparticles treated with acid and p-dimethylaminobenzaldehyde, wherein the core particle size of the upconversion nanoparticles is 25.85-28.85 nm and the shell thickness is 0.98-1.38 nm. The upconversion fluorescent probe uses acid-washed hydrophilic core-shell UCNPs NaYF4:Yb,Tm@NaYF4 as the energy donor and p-dimethylaminobenzaldehyde as the recognition molecule. Isoniazid and p-dimethylaminobenzaldehyde generate a Schiff base under acidic conditions. The 475nm absorption peak of the base overlaps with the emission spectrum of the upconversion nanoparticles, triggering an internal filtration effect that leads to selective quenching of the 475nm fluorescence while the 655nm fluorescence remains unchanged, causing the fluorescence color to change from blue to red, thus achieving dual-wavelength ratio detection.

2. The upconversion fluorescent probe according to claim 1, characterized in that, The pickling process specifically involves ultrasonically washing the upconversion nanoparticles with concentrated acid to change their zeta potential from negative to positive, with the potential change amplitude being >50 mV.

3. The upconversion fluorescent probe according to claim 1, characterized in that, The fluorescence signal fluctuation of the probe is less than 3% within a temperature range of 10-80℃.

4. A method for detecting isoniazid, characterized in that, Includes the following steps: S1. Mix the upconversion fluorescent probe according to any one of claims 1-3 with concentrated acid to obtain a probe solution; S2. Add the whole blood sample to be tested to the probe solution and react for at least 70 seconds to obtain the reaction solution; S3. Detect the fluorescence intensity ratio I between 475 nm and 655 nm of the reaction solution. 475 / I 655 ; S4. The obtained fluorescence intensity is compared with I 475 / I 655 Substitute the values ​​into the pre-calibrated standard curve to calculate the concentration of isoniazid.

5. The method for detecting isoniazid according to claim 4, characterized in that, In step S3, the formula for calculating the standard curve is I. 475 / I 655 = -0.0089C + 0.98; where C is the isoniazid concentration in μm; I 475 Fluorescence intensity at 475 nm; I 655 The fluorescence intensity is at 655 nm.

6. The detection method according to claim 4, characterized in that, Its detection limit is 2.4 μm.

7. A portable isoniazid detection sensor, characterized in that, Including immunochromatographic test strips having an upconversion fluorescent probe as described in any one of claims 1-3.

8. The portable isoniazid detection sensor according to claim 7, characterized in that, Also includes: An optical excitation and signal acquisition module is used to excite upconversion nanoparticles and separate target fluorescence; A cavity for accommodating the optical excitation and signal acquisition module; An image acquisition module and an RGB fluorescence ratio analysis system are used to convert fluorescence color changes into B / R values ​​and calculate concentrations. The RGB fluorescence ratio analysis system establishes a linear equation between the B / R value and the isoniazid concentration, with a detection range of 0-100 μm; the spiked recovery rate of whole blood samples is 98-110%, and the relative standard deviation is ≤3.2%.

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