Up-conversion fluorescence probe, isoniazide detection method and portable isoniazide detection sensor

Abstract

The invention discloses an up-conversion fluorescent probe, an isoniazide detection method and a portable isoniazide 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, the core particle size of the up-conversion nanoparticles ranges from 25.85 nm to 28.85 nm, and the thickness of a shell layer ranges from 0.98 nm to 1.38 nm. The lanthanide doped core-shell structure up-conversion nanoparticles are used as energy donors, p-dimethylaminobenzaldehyde is used as a specific recognition molecule, and ratio-type fluorescence detection is realized through an inner filter effect. Isoniazide reacts with DMAAB to generate Schiff base, so that fluorescence at 475 nm is quenched while fluorescence at 655 nm is unchanged, and the color of the fluorescence is changed from blue to red. Due to the near-infrared excitation characteristic of the up-conversion nanoparticles, the up-conversion nanoparticles are not easily interfered by background fluorescence, so that the rapid quantitative detection of isoniazide in whole blood is realized, and the detection limit is as low as 2.4 mu m. The method is easy and convenient to operate, high in anti-interference performance and suitable for clinical individualized medication monitoring.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, in particular to an up-conversion fluorescent probe, an isoniazid detection method and a portable isoniazid detection sensor. Background Art

[0002] Isoniazid, a first-line anti-tuberculosis drug, has been widely used worldwide due to its highly selective and significant efficacy against Mycobacterium tuberculosis. In clinical practice, isoniazid not only effectively controls tuberculosis symptoms but also offers a high safety profile due to its low toxicity. However, long-term use of isoniazid can 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). Polymorphisms in the N-acetyltransferase 2 (NAT2) gene lead to differences in fast and slow acetylating phenotypes, resulting in a 5-20-fold fluctuation in blood drug concentrations at the same dose. This metabolic heterogeneity directly contributes to treatment failure or adverse drug reactions in some patients. Although isoniazid's efficacy far exceeds that of alternative drugs, the conflict between its toxicity risk and the need for personalized medication highlights the urgency of real-time blood drug concentration monitoring. Current detection technologies such as HPLC and UV spectrophotometry suffer from expensive equipment, complex pretreatment, and the inability to monitor drug concentrations in real time. While upconversion nanoprobes have been used in biological assays, these probes have previously been limited to serum. The detection method proposed in this paper, based on a chemical reaction between upconversion nanoprobes and isoniazid, effectively avoids interference from background fluorescence in whole blood and can be used to measure 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 raised in the above background technology. The fluorescent probe based on upconversion nanoparticles and p-dimethylaminobenzaldehyde is used for highly sensitive detection of isoniazid concentration in whole blood.

[0004] To achieve the above object, the present invention provides the following technical solutions: 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.

[0005] As a further solution of the present invention: the pickling treatment specifically comprises: ultrasonic pickling the upconversion nanoparticles with concentrated acid to change their zeta potential value from negative to positive, and the potential change amplitude is >50 mV.

[0006] As a further solution of the present invention: the fluorescence signal fluctuation of the probe is less than 3% within the temperature range of 10-80°C.

[0007] In a second aspect, the present invention discloses a method for detecting isoniazid, comprising the following steps: S1, mixing the above-mentioned upconversion fluorescent probe with concentrated acid to obtain a probe solution; S2. adding the whole blood sample to be tested to the probe solution and reacting for at least 70 seconds to obtain a reaction solution; S3, detecting the fluorescence intensity ratio of the reaction solution at 475 nm to 655 nm. 475 / I 655 ; S4, the obtained fluorescence intensity ratio I 475 / I 655 Substitute into the standard curve and calculate the concentration of isoniazid.

[0008] As a further solution of the present invention: in step S3, the calculation formula of the standard curve is: 475 / I 655 = -0.0089C + 0.98; where C is the isoniazid concentration in μm; I 475 is the fluorescence intensity at 475 nm; I 655 is the fluorescence intensity at 655 nm.

[0009] As a further solution of the present invention: its detection limit is 2.4 μm.

[0010] In a third aspect, the present invention discloses a portable isoniazid detection sensor, comprising an immunochromatographic test strip having the above-mentioned upconversion fluorescent probe.

[0011] Furthermore, the portable isoniazid detection sensor also includes: Optical excitation and signal acquisition module, used to excite upconversion nanoparticles and separate target fluorescence; A cavity, used to accommodate the optical excitation and signal acquisition module; 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.

[0012] The optical excitation and signal acquisition module includes a 980 nm laser, an optical filter and a cuvette holder.

[0013] As a further embodiment of the present invention: The RGB fluorescence ratio analysis system establishes a linear equation between the B / R value and the isoniazid concentration. The detection range of the portable isoniazid detection sensor is 0-100 μm. The recovery rate of spiked whole blood samples is 98-110%, and the relative standard deviation is ≤3.2%.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses acid-washed, hydrophilized core-shell UCNPs (NaYF4:Yb,Tm@NaYF4) as an energy donor and p-dimethylaminobenzaldehyde (DMAAB) as a recognition molecule. Under acidic conditions, isoniazid and DMAAB form a Schiff base. Its 475 nm absorption peak overlaps with the emission spectrum of the upconversion nanoparticles (UCNPs), triggering an inner filter effect (IFE) that selectively quenches the 475 nm fluorescence. The 655 nm fluorescence remains unchanged, shifting the fluorescence color from blue to red, enabling dual-wavelength ratiometric detection.

[0015] 2. The probe prepared by this invention can be used to detect isoniazid concentrations in whole blood. Due to the near-infrared excitation characteristics of upconversion nanoparticles, they are less susceptible to interference from background fluorescence, 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 and highly resistant to interference, making it suitable for clinical personalized drug monitoring.

[0016] 4. Rapid response within 70 seconds, recovery rate of spiked whole blood is 98-110%, RSD≤3.2%, and it has the characteristics of wide detection temperature range (10-80℃) and excellent pH stability.

[0017] 5. The present invention combines upconversion nanoprobes with immunochromatographic test strips to construct a portable detection platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 TEM image and particle size characterization of the upconversion nanoparticles prepared in Example 1; Figure 2 XRD, XPS and zeta potential spectra of the upconversion nanoparticles prepared in Example 1; Figure 3 FTIR spectrum of the material prepared in Example 1 of the present invention; Figure 4 This is a performance test of the sensitivity, selectivity and anti-interference of the probe in Example 3 of the present invention; Figure 5 The stability test of the upconversion probe prepared in Example 3 of the present invention in the temperature range of 10-80°C is performed; Figure 6 The stability test of the upconversion probe prepared in Example 3 of the present invention within 140 minutes; Figure 7 The reaction efficiency of the upconversion probe prepared in Example 3 of the present invention with isoniazid at different pH values ​​was determined, and the acidic condition (pH value of 1) was determined to be the optimal reaction environment; Figure 8 This is the response time curve of the probe prepared in Example 3 of the present invention, showing that the signal reaches a plateau after 70 seconds; the abscissa is time, and the ordinate is the absorbance value of the probe; Figure 9 Schematic diagram of the probe prepared in Example 4 of the present invention for detecting the concentration of isoniazid in whole blood; Figure 10 The effect of the probe prepared in Example 4 of the present invention in whole blood detection: Figure 10 A and Figure 10 B shows the relationship between the fluorescence color gradient and concentration. Figure 10 C shows that the B / R value is linearly related to the concentration. Figure 10 D showed spike recoveries of 101.59-109.5%; Figure 11 This is a schematic diagram of the structure of the portable isoniazid detection sensor in the present invention, wherein Figure 11 A is a specific component of the sensor device, Figure 11 B is the overall structure of the sensor device. DETAILED DESCRIPTION

[0019] To facilitate understanding of the present invention, the present invention will be described more fully below in conjunction with specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0021] Example 1: Preparation of core-shell UCNPs 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. The mixture was then reacted at 300°C for 1 hour to obtain upconversion nanoparticles (UCNPs).

[0022] 2. Shell coating: The core solution and YCl3 precursor are mixed in a ratio of 1:0.2 and coated with NaYF4 inert shell in the same way.

[0023] 3. Pickling treatment: pH = 1 hydrochloric acid ultrasonic for 1 hour, centrifuge and then disperse in water ( Figure 2 B verifies the zeta potential shift).

[0024] Example 2: Preparation of upconversion fluorescent probe The probe solution was prepared by adding 50 μL of acid-washed UCNPs 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. Ethanol was added to the probe solution to adjust the final volume to 2 mL. Equal volumes of isoniazid solutions of varying concentrations were then introduced into the probe solution and mixed thoroughly. 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.

[0025] Example 3: Probe performance test The pH of the probe system was adjusted using hydrochloric acid and sodium hydroxide, and the reaction degree between the probe and isoniazid was evaluated under different pH conditions to determine the optimal pH value. The results are shown in Figure 7 In addition, the thermal stability of the probe was evaluated in the temperature range of 10-80 ℃ to investigate the effect of temperature on its performance. Figure 5 .

[0026] First, prepare the isoniazid standard solution (solvent is water). At room temperature, mix 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μM isoniazid standard solutions with 2 mL of the probe solution. Record the test fluorescence spectra and linearly fit the isoniazid concentration C and I. 475 / I 655 , and obtain the standard curve I 475 / I 655 = -0.0089C + 0.98.

[0027] The probe performance test results are as follows: 1. Sensitivity: I 475 / I 655 Linear correlation with 0-100 μM isoniazid (e.g. Figure 4 C).

[0028] 2. Specificity: Signal change <5% at 100 μM interfering substances (ascorbic acid / dopamine, etc.) (e.g. Figure 4 D).

[0029] 3. Stability: Signal fluctuation <3% within 10-80℃ and 140 minutes (e.g. Figure 5 and Figure 6 shown).

[0030] 4. Response time <70 seconds, supporting real-time monitoring.

[0031] Example 4: Whole blood spiked with isoniazid detection 1. Take whole blood from a healthy individual and spike it with isoniazid (concentration gradient range 10-70 μM), add 2 ml of the probe solution obtained in Example 2 and react for 70 seconds.

[0032] 2. The B / R value was measured on the smartphone platform and the recovery rate was calculated to be 101.59-109.5% (e.g. Figure 10 D).

[0033] 3. Compare the above isoniazid concentration results with those of HPLC detection. The relative error between the two is less than 5% (e.g. Figure 10 C).

[0034] Example 5: Portable isoniazid detection sensor like Figure 11 As shown, this embodiment discloses a portable isoniazid detection sensor. Figure 11 A, the portable isoniazid detection sensor includes the following components: Immunochromatographic test strips with upconversion fluorescent probes (i.e. Figure 11 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 allowed to dry naturally.

[0035] Optical excitation and signal acquisition module (i.e. Figure 11 The 980 nm laser in A) is used to excite the upconversion nanoparticles and isolate the target fluorescence; A cavity, used to form a dark chamber and accommodate the optical excitation and signal acquisition module and the test strip; Image acquisition module and RGB fluorescence ratio analysis system (built in Figure 11 The smartphone in A is used to convert the acquired fluorescence color changes into B / R values ​​and calculate concentration. This image acquisition module and RGB fluorescence ratio analysis system can be implemented on a smartphone or tablet. The camera captures the original image, and the optical filter is physically installed in front of the camera. The optical excitation and signal acquisition module includes a 980 nm laser, an optical filter, and a cuvette holder.

[0036] See also Figure 11 B, shows the overall structure of the assembled portable isoniazid detection sensor.

[0037] During the test, the sample is added to the test strip, which is then inserted into the cavity and closed to create a completely black detection environment. After 70 seconds, the camera is turned on to photograph the test strip in the dark room, and the RGB fluorescence ratio analysis system is used to convert the fluorescence color changes of the collected image into a B / R value to calculate the isoniazid concentration in the sample.

[0038] The materials prepared in the examples of the present invention and the performance tests were characterized, as described in detail below: Figure 1 TEM images and particle size characterization of the upconversion nanoparticles prepared in Example 1, wherein: Figure 1 A and Figure 1 B is the transmission electron microscopy (TEM) morphology of the core and core-shell upconversion nanoparticles, indicating that the synthesized nanoparticles have uniform morphological characteristics. Figure 1 C is the lattice fringe image of the core-shell upconversion nanoparticles under high-resolution transmission electron microscopy (HRTEM), which confirms the excellent crystalline quality of the nanoparticles. Clear lattice fringes can be observed, which are completely consistent with the standard crystal plane characteristics. Figure 1 D is the elemental analysis diagram of core-shell upconversion nanoparticles. Figure 1 E and Figure 1 F is the particle size distribution of 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, indicating that a uniform inert shell with a thickness of about 1.18 nm is formed. The particle size distribution is narrow, showing good size consistency and controllability.

[0039] Figure 2 The XRD, XPS and zeta potential spectra of the upconversion nanoparticles prepared in Example 1 are as follows: Figure 2 Figure A shows the X-ray diffraction analysis of the core and core-shell upconversion nanoparticles corresponding to the standard card. By comparing the XRD diffraction peaks of the particle core and core-shell structure with the standard card (16-0334), it is confirmed that the synthesized particles have a standard hexagonal phase structure. Figure 2 B is the zeta potential analysis diagram of the core-shell nanoparticles before and after acid washing. The zeta potential of the particles before acid washing is -15.38 mV, while the zeta potential after acid washing jumps to 37.6 mV, indicating that the particle surface changes from negative charge to positive charge, confirming 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.

[0040] 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 pickling, 2927 cm -1 and 2855 cm -1 The absorption peaks are attributed to the stretching vibration of the methylene group in the oleic acid ligand, and the rose-red line corresponds to the infrared spectrum of the particles after acid washing. These characteristic absorption peaks disappear significantly, indicating that the oleic acid ligand has been successfully removed.

[0041] Figure 4 This is a performance test of the sensitivity, selectivity and anti-interference of the probe in Example 3 of the present invention. Figure 4 A and Figure 4 B shows the gradient change of the fluorescence intensity and absorbance of the upconversion nanoprobe with the addition of isoniazid. Under the condition of fixed probe concentration, as the concentration of isoniazid increases (0-100 μM), the upconversion fluorescence intensity at 475 nm shows concentration-dependent quenching, while the fluorescence at 655 nm remains stable. This specific response forms a unique ratiometric signal (I 475 / I 655 ). Figure 4 C is the ratio of the fluorescence intensity of the probe and the linear relationship between the absorbance value and the isoniazid concentration. Both modes have good linear relationships. Figure 4 D is the selectivity and anti-interference value of the probe. Even in the presence of multiple interferents, the probe can still efficiently detect isoniazid and will not be significantly interfered with by other components.

[0042] Figure 5 The stability of the upconversion probe prepared in Example 3 of the present invention at different temperatures. Figure 5 A is the relationship curve between the fluorescence intensity and wavelength of the upconversion fluorescence probe, Figure 5 B is the curve of the fluorescence intensity of the upconversion fluorescence probe changing with temperature. Figure 5 C is the relationship curve between the ultraviolet absorption value of the up-conversion fluorescent probe and the wavelength, Figure 5 D is the temperature-dependent curve of the UV absorbance of the upconversion fluorescent probe. As the temperature gradually increases from 10°C to 80°C, the probe's fluorescence intensity and UV absorbance change little, with no significant fluctuation or attenuation, indicating the probe's good stability under varying temperatures. This result demonstrates that the probe maintains its performance over a wide temperature range.

[0043] Figure 6 The stability of the upconversion probe prepared in Example 3 of the present invention at different times; wherein: Figure 6 A is the relationship curve between the fluorescence intensity and wavelength of the upconversion fluorescence probe, Figure 6B is the curve of the fluorescence intensity of the upconversion fluorescent probe changing with time, Figure 6 C is the relationship curve between the ultraviolet absorption value of the up-conversion fluorescent probe and the wavelength, Figure 6 D is a plot of the UV absorbance of the upconversion fluorescent probe over time. The probe exhibited no significant changes in fluorescence intensity or UV absorbance from 0 to 140 minutes, demonstrating that the probe maintains relatively stable performance over extended periods. This result further validates the probe's long-term stability, ensuring reliable detection even under extended exposure to reaction conditions during actual testing.

[0044] Figure 7 The stability of the upconversion probe prepared in Example 3 of the present invention at different pH values. As can be seen from the figure, the probe has a faster reaction rate and higher reaction efficiency in an acidic environment. Acidic environments can effectively promote the nucleophilic reaction between isoniazid and p-dimethylaminobenzaldehyde, thereby accelerating the formation of Schiff bases. Therefore, rationally adjusting the pH value of the reaction system is key to achieving probe sensitivity and accuracy.

[0045] Figure 8 This is the optimal reaction time for preparing the probe in Example 3 of the present invention. As shown in the figure, the reaction system reaches a stable state after about 70 seconds of reaction between isoniazid and p-dimethylaminobenzaldehyde. This means that the probe can complete the reaction in a short time, and any fluctuations during the reaction can be quickly stabilized, ensuring the 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.

[0046] 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. The probe solution is first loaded into the absorbent pad and combined with the backing plate to form the detection area. Subsequently, the constructed immunochromatographic device was combined with existing 3D printing technology to develop a portable sensing device (such as Figure 11 The device is specifically designed for rapid detection of isoniazid in complex environments, making on-site detection of isoniazid more efficient and reliable.

[0047] Figure 10 This is the effect of the probe prepared in Example 4 of the present invention in whole blood detection. Figure 10 A and Figure 10 B shows the correspondence between the isoniazid concentration gradient and the change in probe fluorescence color after spiking experiments with different concentrations of isoniazid, as well as the division of clinical treatment effects and dangerous concentration intervals. Figure 10C represents the relationship between the spiked amount of isoniazid at different concentrations and the B / R ratio. The data show that the B / R ratio changes significantly with increasing isoniazid concentration, exhibiting a good linear relationship within a certain range. Furthermore, when the sample color detected by the sensing platform shifts red, it indicates that the isoniazid concentration in whole blood has approached or reached a dangerous level, prompting caution. This phenomenon not only verifies the sensitivity and accuracy of the sensing platform but also demonstrates its effectiveness in practical applications. Figure 10 D is the result of the probe spike recovery experiment in fluorescence mode. When 10μM, 30μM, 50μM and 70μM isoniazid were added, the sample recoveries were 109.5%, 100.4%, 100.04% and 101.59%, respectively, and the relative standard deviation remained between 2.5% and 3.2%, indicating that the detection results of this platform in actual samples have high accuracy and reliability.

[0048] Figure 11 This is a schematic diagram of the structure of a portable isoniazid detection sensor. Figure 11 A shows the specific components of the sensor structure. Figure 11 B is a schematic diagram of the structural assembly of the portable isoniazid detection sensor before testing.

[0049] Although this specification is described according to implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0050] Therefore, the above description is only a preferred embodiment of the present application and is not intended to limit the scope of implementation of the present application; that is, all equivalent modifications made according to the scope of the claims of the present application are within the scope of protection of the claims of the present application.

Claims

1. An upconversion fluorescent probe, characterized in that The invention is composed of acid-washed lanthanide-doped core-shell 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.

2. The upconversion fluorescent probe according to claim 1, characterized in that The acid washing treatment specifically includes: ultrasonically washing the upconversion nanoparticles with concentrated acid to change their zeta potential value from negative to positive, and the potential change amplitude is greater than 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 the temperature range of 10-80°C.

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

5. The method for detecting isoniazid according to claim 4, wherein In step S3, the calculation formula of the standard curve is: 475 / I 655 = -0.0089C + 0.98; where C is the isoniazid concentration in μm; I 475 is the fluorescence intensity at 475 nm; I 655 is the fluorescence intensity 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: The invention comprises an immunochromatographic test strip having the up-conversion fluorescent probe according to any one of claims 1 to 3.

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

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