Miniature device for rapidly and directly detecting poison sample and use method thereof

Abstract

The invention discloses a miniature device for rapidly and directly detecting a poison sample and a use method of the miniature device, and relates to the technical field of analytical chemistry and biosensors.The miniature device comprises a magnetic enrichment unit, a magnetic recognition probe and a signal probe. A magnetic enrichment unit is utilized to enrich a magnetic recognition probe combined with a target object in a micro-droplet at the top end, then a signal probe is added, qualitative and quantitative analysis of the target object is achieved by detecting a generated fluorescence signal, and the specific magnetic recognition probe does not need sample pretreatment; the magnetic recognition probe and the signal probe are combined to realize ultrahigh-sensitivity detection; all detection steps are integrated in the micro-droplet at the top end of the magnetic bar, so that the device is extremely miniaturized and portable, reagent consumption and cost are reduced, the device solves the problems of complex sample pretreatment, serious matrix interference and the like, and miniaturized and low-cost field rapid detection of high-sensitivity and high-specificity detection is realized.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry and biosensor technology, and in particular to a miniaturized device and method for rapid, on-site detection of target molecules such as drugs and poisons. Background Art

[0002] The rapid and accurate detection of various samples, especially new drugs (such as nitrazepam), is a key step in combating drug crime and ensuring public safety. Currently, mainstream methods for drug detection include spectral analysis, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). However, these traditional technologies have many limitations in practical applications: (1) Sample pretreatment is complex and time-consuming: Existing detection methods usually require tedious pretreatment steps such as sample extraction and purification. The entire process takes several hours or even days, which is not conducive to rapid on-site investigation.

[0003] (2) High-precision technology relies on laboratory environment: Although chromatography-mass spectrometry is accurate, the equipment is expensive and bulky, requiring professional personnel to operate, and the sample pretreatment process is complicated (such as solid phase extraction and derivatization). The detection cycle is as long as several hours, and the single cost is high, which cannot meet the needs of rapid on-site screening.

[0004] (3) Inadequate performance of rapid detection technologies: Although immunoassays (such as immunochromatographic test strips) are rapid, their core antibodies are susceptible to interference from proteins and lipids in complex biological samples such as blood and urine, resulting in a high false positive rate (up to 15%) and limited sensitivity (the detection limit is usually greater than 5 ng / mL). Spectroscopic techniques have low signal-to-noise ratios and insufficient sensitivity (>1 μg / mL) when directly detecting complex matrix samples. Electrochemical sensors are susceptible to electrode contamination, resulting in signal drift and poor qualitative analysis.

[0005] (4) Existing improvement plans fail to resolve the core bottleneck: Although some studies have attempted to improve the method through technologies such as nanomaterials, microfluidic chips or aptamer sensors, they have not yet effectively resolved the core contradictions such as matrix interference of complex biological samples, the coordinated stability of probe signals, and the simplification of detection processes.

[0006] Therefore, how to provide a miniature device for rapid and direct detection of poison samples and its use method, solve the problem of complex sample pretreatment, resist matrix interference, and achieve high sensitivity and high specificity detection of miniaturized, low-cost on-site rapid detection equipment, is a technical problem that needs to be solved urgently by technical personnel in this field. Summary of the Invention

[0007] The purpose of the present invention is to provide a micro device for rapid and direct detection of poison samples and its use method, which solves the problem of complex sample pretreatment, can resist matrix interference, and can achieve miniaturized, low-cost on-site rapid detection with high sensitivity and high specificity.

[0008] To achieve the above object, the present invention provides the following solutions: The present invention discloses a micro device for quickly and directly detecting poison samples and a method for using the same, comprising: A magnetic enrichment unit, the magnetic enrichment unit consisting of a magnetic rod and a pipette tip fixed to the end thereof; A magnetic recognition probe, comprising a functionalized magnetic nanosphere coated with a layer of polydopamine covalently linked to an aptamer capable of specifically binding to a target poison; The signal probe is a vesicle structure.

[0009] Preferably, the interior of the pipette tip is filled with a gel medium.

[0010] Preferably, the structure of the magnetic nanospheres is based on Fe3O4 magnetic nanospheres as the core.

[0011] Preferably, the aptamer is a DNA aptamer that can specifically bind to the target poison molecule.

[0012] Preferably, the signal probe is a fluorescent signal probe.

[0013] Preferably, the fluorescent signal probe is assembled from polydiacetylene and phospholipid.

[0014] The present invention provides a method for using a micro-device for quickly and directly detecting poison samples, comprising the following steps: S1, mixing a biological sample to be tested (such as blood or urine) without any pretreatment with the magnetic recognition probe solution; S2, immersing the end of the pipette tip of the magnetic enrichment unit into the mixed sample solution to form a reaction micro-zone; S3, adding the fluorescent signal probe solution to the droplets in the reaction micro-zone; S4, detecting the presence or absence of a fluorescent signal for qualitative judgment, and performing quantitative analysis by detecting the intensity of the fluorescent signal.

[0015] Compared with the prior art, the present invention has achieved the following technical effects: The present invention discloses a micro-device for rapid and direct detection of poison samples and its use method. Through specific magnetic recognition probes, target poisons can be directly captured from complex biological matrices such as blood and urine, allowing direct detection without sample pretreatment. The magnetic recognition probes are combined with signal probes to increase the probability of collision between the target and the fluorescent probe, achieving ultra-high sensitivity detection. All detection steps are integrated into a tiny droplet at the tip of the magnetic rod, making the device extremely miniaturized and portable, reducing reagent consumption and costs, and also possessing strong universality. This device solves the problem of complex sample pretreatment and is resistant to matrix interference. It is a miniaturized, low-cost, on-site rapid detection device that can achieve high sensitivity and high specificity detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a schematic diagram of the preparation principle of a micro-device for rapid and direct detection of poison samples and its use method in this embodiment; Figure 2 This is a schematic diagram of the preparation process of a magnetic tip microdevice for a microdevice for rapid and direct detection of poison samples and its use method in this embodiment; Figure 3 This is a schematic diagram of a micro-device for rapid and direct detection of poison samples and its use method, and the preparation of artificial cell-specific probes and the principle of signal generation; Figure 4 This is a schematic diagram of the experimental results of a poison sample of a micro device for rapid and direct detection of poison samples and its use method in this embodiment; Figure 5 This is a schematic diagram of a micro-device for rapid and direct detection of poison samples and its use method for direct blood detection in this embodiment; Explanation of reference numerals: 1 magnetic rod; 2 agarose gel; 3 pipette tip; 4 magnetic enrichment unit. DETAILED DESCRIPTION

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0019] The purpose of the present invention is to provide a miniature device for rapid and direct detection of poison samples and its use method, so as to solve the problem of complex sample pretreatment, resist matrix interference, and realize high-sensitivity and high-specificity detection of miniaturized, low-cost on-site rapid detection equipment.

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] like Figure 1 As shown, this embodiment provides a micro device for rapid and direct detection of poison samples and a method for using the same, including: A magnetic enrichment unit 4, which is composed of a magnetic rod 1 and a pipette tip 3 fixed to the end thereof; A magnetic recognition probe, comprising a functionalized magnetic nanosphere coated with a layer of polydopamine covalently linked to an aptamer capable of specifically binding to a target poison; The signal probe is a vesicle structure.

[0022] The interior of the pipette tip is filled with agarose gel 2.

[0023] The method of using the micro-device for rapid and direct detection of poison samples of the present invention is as follows: S1, mixing a biological sample to be tested (such as blood, urine) without any pretreatment with the magnetic recognition probe solution, so that the aptamer on the magnetic recognition probe specifically binds to the target poison molecule in the sample.

[0024] S2, immerse the end of the pipette tip 3 of the magnetic enrichment unit 4 into the mixed sample solution, and the magnetic recognition probe bound to the target poison is adsorbed and enriched into the micro-buffer droplet at the top of the pipette tip 3 by the magnetic force of the magnetic rod 1 to form a reaction micro-area.

[0025] S3, adding the fluorescent signal probe solution into the droplets in the reaction micro-area.

[0026] S4, qualitative judgment is made by detecting the presence or absence of fluorescent signals, and quantitative analysis is performed by detecting the intensity of fluorescent signals. The fluorescence intensity is positively correlated with the concentration of the poison in the sample to be tested.

[0027] Example 1 like Figure 4 As shown in the figure, by adding different concentrations of poison samples under the same conditions, fluorescence measurement is performed. The fluorescence graph shows that as the concentration increases, the fluorescence increases linearly, which proves that the detection method is feasible.

[0028] Example 2 Compared with the prior art, the present invention has the following technical effects:

[0029] Example 3 Polydopamine-coated vesicles / Fe₃O₄ nanospheres (Fe₃O₄@PDA), a GST-attached aptamer (GST-Aptamer / miRNA-Aptamer), and a hairpin probe targeting the target miRNA were added to the tip of magnetic rod 1 in a single-drop magnetic microreactor. The microreactor was then inserted into the actual sample, and GST and miRNA-196a were simultaneously extracted from the sample solution. As the pH in the SDMR system shifted from slightly acidic to slightly alkaline, GST and GST-Aptamer were released from the nanospheres and captured by the polydiacetylene vesicles, resulting in a highly enhanced fluorescence signal, enabling the detection of GST levels. Simultaneously, the target miRNA was selectively recognized by the hairpin probe and triggered a DNA amplification reaction, constructing a DNA strand with two independent probes: the G-four-plex / TAIN and Cy5. Due to the spatial confinement of the single-drop system, the G-four-plex / TAIN was enriched, resulting in a highly enhanced fluorescence signal, enabling sensitive detection of the miRNA.

[0030] like Figure 5 As shown, this invention can simultaneously detect GST and miRNA-196a in urine and blood without sample pretreatment, achieving enrichment of the target species while reducing interference from matrix effects. This improves upon existing methods of single-protein detection by enabling direct processing and analysis of complex samples through microextraction in a liquid-liquid single-drop system. Furthermore, this technology can simultaneously detect biomarkers—proteins and miRNAs—in diverse disease states, providing a rapid, sensitive, and accurate predictive diagnostic tool.

[0031] This specification uses specific examples to illustrate the principles and implementation methods of the present invention. The above examples are only intended to help understand the method and core concept of the present invention. At the same time, those skilled in the art will find that the specific implementation methods and application scopes may vary based on the concept of the present invention. In summary, the contents of this specification should not be construed as limiting the present invention.

Claims

1. A micro device for rapid and direct detection of poison samples and a method for using the same, characterized in that: include: A magnetic enrichment unit, the magnetic enrichment unit consisting of a magnetic rod and a pipette tip fixed to the end thereof; A magnetic recognition probe, comprising a functionalized magnetic nanosphere coated with a layer of polydopamine covalently linked to an aptamer capable of specifically binding to a target poison; The signal probe is a vesicle structure.

2. A micro device for rapid and direct detection of poison samples and a method for using the same according to claim 1, characterized in that: The interior of the pipette tip is filled with gel medium.

3. The micro device for rapid and direct detection of poison samples and the method for using the same according to claim 1, characterized in that: The structure of the magnetic nanospheres is based on Fe3O4 magnetic nanospheres as the core.

4. The micro device for rapid and direct detection of poison samples and the method for using the same according to claim 1, characterized in that: The aptamer is a DNA aptamer that can specifically bind to the target poison molecule.

5. The micro device for rapid and direct detection of poison samples and the method for using the same according to claim 1, characterized in that: The signal probe is a fluorescent signal probe.

6. The micro device for rapid and direct detection of poison samples and the method for using the same according to claim 5, characterized in that: The fluorescent signal probe is formed by assembling polydiacetylene and phospholipid.

7. The micro device for rapid and direct detection of poison samples and the method for using the same according to claim 1, characterized in that: The method comprises the following steps: S1, mixing a biological sample to be tested (such as blood or urine) without any pretreatment with the magnetic recognition probe solution; S2, immersing the end of the pipette tip of the magnetic enrichment unit into the mixed sample solution to form a reaction micro-zone; S3, adding the fluorescent signal probe solution to the droplets in the reaction micro-zone; S4, detecting the presence or absence of a fluorescent signal for qualitative judgment, and performing quantitative analysis by detecting the intensity of the fluorescent signal.

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