Single-molecule electric sensor for detecting content of iodide ions in human urine and detection method

Abstract

The invention discloses a single-molecule electric sensor and a detection method for detecting the content of iodide ions in human urine, the detection method is based on a scanning tunnel microscopy break junction method, and the detection process comprises the following steps: in a solution containing iodide ions of a to-be-detected object, adding a detection reagent, the method comprises the following steps: controlling an Au needle tip to approach a probe molecule modified Au substrate through piezoelectric ceramics until a preset needle tip current value is 50nA, and then controlling the needle tip to continuously extend forwards towards the substrate by a certain distance under an additionally applied pulse voltage, so as to ensure that the needle tip is in contact with the substrate; controlling the needle tip to be far away from the substrate at a constant speed, forming a metal-probe molecule-metal junction in the process, and recording a conductance-distance curve of the needle tip at the same time; the process is repeated, thousands of conductance-distance curves are counted to obtain a one-dimensional conductance diagram, Gaussian fitting is carried out on conductance peaks in the one-dimensional conductance diagram to obtain the area of a single-molecule conductance peak, and quantitative detection can be carried out on the content of iodide ions in the solution to be detected based on the area of the single-molecule conductance peak.

Description

Technical Field

[0001] The present invention relates to the field of molecular electronic technology, and in particular to a single-molecule electrical sensor for detecting the iodide ion content in human urine and a detection method. Background Art

[0002] Iodine is an essential substance for the synthesis of thyroid hormone and an essential trace element for the human body. Iodine promotes oxidation, participates in regulating body temperature, and maintains normal metabolism and life activities. Excessive or insufficient iodine intake can cause certain harm to the human body. Therefore, it is of great significance to detect the content of iodine ions. At present, the methods for detecting iodine ions include fluorescence, colorimetry, ion-selective electrode method, etc. However, these methods have certain limitations. For example, optical methods require fluorescent labels, and traditional electrical methods are affected by time resolution and low signal-to-noise ratio. On the other hand, halogen ions can be specifically adsorbed on metal surfaces, which may affect molecular adsorption and change the properties of single-molecule junctions, thus providing a unique opportunity to detect iodide at the single-molecule level. Single-molecule electrical detection can achieve direct, real-time and label-free monitoring of dynamic processes of chemical reactions or bioengineering at the single-molecule level. This provides an ideal platform for exploring detailed information of chemical and biological reactions. For example, single-molecule detection technology represented by scanning tunneling microscopy has powerful sensing capabilities, and the collected single-molecule data is proportional to the concentration of the analyte and is sensitive to subtle changes in the chemical composition of species within the molecule. Based on the above background, we invented a single-molecule electrical sensor with high sensitivity and selectivity, which can quantitatively detect iodide ions in solution without labeling. It can also be actually applied to human urine for detection, opening up a new way to utilize single-molecule electrical detection. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a single molecule electrical sensor for detecting the iodine ion content in human urine, comprising:

[0004] A scanning tunneling microscope, a needle tip, a metal substrate, an electrolytic cell and an O-ring; the needle tip is inserted into the scanning head of the scanning tunneling microscope, the metal substrate is installed in the electrolytic cell, and the O-ring is arranged on the outside of the bottom of the electrolytic cell.

[0005] Optionally, the metal substrate further comprises gold balls for providing atomically flat gold single crystal electrodes for adsorbing the molecules to be detected.

[0006] The present invention also discloses a detection method of a single-molecule electrical sensor for detecting the iodine ion content in human urine, comprising: controlling a needle tip to approach a metal substrate through a piezoelectric ceramic, collecting a current value during the approaching process, and when the current value reaches a preset current value, the piezoelectric ceramic controls the needle tip to approach a preset distance in the direction of the substrate, and when the single-molecule electrical sensor detects the current value, the piezoelectric ceramic controls the needle tip to move away from the metal substrate at a constant speed;

[0007] When the molecules in the electrolytic cell solution that have anchoring groups and can react with metals form a metal-molecule-metal junction between the needle tip and the metal substrate;

[0008] When the needle tip is pulled upward, the connection between the molecule and the electrode is disconnected, completing a cleavage process;

[0009] Collect the tunnel current during the splitting process to obtain the conductance-distance curve;

[0010] The obtained conductivity-distance curve is plotted as a one-dimensional conductivity map, and the single-molecule conductivity value is obtained after Gaussian fitting of the conductivity peak in the one-dimensional conductivity map. The detection result of the iodide ion content in human urine is obtained based on the single-molecule conductivity peak area.

[0011] Optionally, the preset range of the current value is 8nA to 80nA.

[0012] Optionally, the needle tip is a gold wire with a length of 1.5 cm, a purity greater than or equal to 99.999%, and Φ=0.25 mm.

[0013] Optionally, hot melt adhesive is evenly coated on an electric soldering iron at 175° C. to insulate and encapsulate the needle tip.

[0014] Optionally, the metal substrate is a gold sheet with a thickness of 0.5 cm and a size of 10×15 mm.

[0015] Optionally, the material of the electrolytic cell is polytetrafluoroethylene.

[0016] Optionally, the process of obtaining the detection result of the iodine ion content in human urine based on the single molecule conductivity value specifically includes:

[0017] 4,4'-bipyridine was added to urine for conductivity measurement, the conductivity peak in the conductivity measurement result was Gaussian fitted to obtain the peak area, the single molecule conductivity peak area was brought into the working curve to obtain the average iodine content in urine.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention takes STM-BJ technology as the core, requires only a small amount of sample, does not require any labeling, has a resolution of up to single molecule level, has high sensitivity and selectivity, is not affected by interfering molecules and interfering ions, and can be actually applied to the detection of iodine ion content in human urine, which is consistent with the clinical method As 3+ -Ce 4+ The average difference of catalytic spectrophotometry is 0.9%, and the detection time is shortened to about 15 minutes. This invention provides an important idea for realizing chemical / biological sensors or detectors with single molecule sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solution of the present invention, 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 creative labor.

[0021] Figure 1 This is a schematic diagram of the structure of a sensor according to an embodiment of the present invention;

[0022] Figure 2 The one-dimensional conductivity statistics of 4,4'-bipyridine in different iodide ion concentrations in the presence of interfering ions and interfering molecules in the embodiments of the present invention are shown;

[0023] Figure 3 The one-dimensional conductivity statistical graph of 4,4'-bipyridine when different concentrations of iodine ions are added to artificial urine according to an embodiment of the present invention;

[0024] Figure 4 This is a graph showing the relationship between the conductivity peak area of ​​4,4'-bipyridine and the iodide ion concentration when different concentrations of iodide ions are added to artificial urine according to an embodiment of the present invention;

[0025] Figure 5 The one-dimensional conductivity diagram of three parallel experiments after adding 4,4'-bipyridine to artificial urine with unknown iodide ion concentration in the embodiment of the present invention;

[0026] Figure 6 The figure is a flow chart of the detection method of the embodiment of the present invention. DETAILED DESCRIPTION

[0027] 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.

[0028] Embodiment 1

[0029] A single-molecule electrical sensor for detecting the iodide ion content in human urine, such as Figure 1 As shown,

[0030] Scanning tunneling microscope, needle tip, metal substrate, electrolytic cell and O-ring; the needle tip is inserted into the scanning head of the scanning tunneling microscope, the metal substrate is installed in the electrolytic cell, and the O-ring is arranged on the outside of the bottom of the electrolytic cell. The O-ring is used to enhance the sealing of the electrolytic cell and prevent electrolyte leakage. The gold ball is part of the metal substrate, and the material is Au. The gold ball is used to provide an atomically flat Au (111) single crystal electrode, which is easy to be adsorbed by the molecule to be measured.

[0031] Embodiment 2

[0032] A detection method of a single molecule electrical sensor for detecting the iodide ion content in human urine, such as Figure 6 As shown, the method includes:

[0033] The piezoelectric ceramics in the scanning tunneling microscope control the needle tip to approach the metal substrate, collect the current value during the approach process, and when the current value reaches the preset value, the piezoelectric ceramic controls the needle tip to approach the substrate to a certain distance. When the instrument detects the current value, the piezoelectric ceramic will control the needle tip to move away from the metal substrate at a constant speed of 20nm / s, and record the conductivity-distance curve. The preparation and installation process of the needle tip specifically includes: before cutting, use a cotton ball dipped in alcohol to clean the tweezers, scissors and the box containing the needle tip, and blow dry with nitrogen. Then take about 1.5cm of gold wire with a purity of ≥99.999% and Φ=0.25mm, first straighten the gold wire with flat tweezers, then clamp the gold wire firmly, place the scissors on the gold wire at an appropriate angle, cut it cleanly after preparation, and quickly remove the scissors. Clamp the needle tip with sharp tweezers and put it in the needle tip box, observe under a microscope, and generally the needle tip suitable for the experiment has a smooth cut surface and a sharp and firm tip. After the needle tip is prepared, hot melt adhesive (polymethyl methyl styrene) is evenly applied on a soldering iron set at 175°C to insulate and encapsulate the needle tip to reduce the interference of the Faraday current. Finally, the encapsulated needle tip is inserted into the STM scanning head, and the tightness of the insertion must be ensured to be appropriate.

[0034] The preparation and treatment process of the metal substrate includes: melting the gold wire in a crucible by hydrogen-oxygen flame, wrapping it with filter paper after cooling, and then repeatedly hitting it with a hammer to form a thin sheet with a thickness of about 0.5 cm. Use scissors to cut a 10×15mm gold sheet as the substrate for fixing the gold ball. Then take a gold wire (purity ≥99.999%, Φ=0.5mm), use the Clavilier method to prepare the single crystal electrode Au(111), after preparation, select a better surface by microscope observation and fix it on the gold substrate with a butane gun. Before each experiment, the Au(111) electrode needs to be immersed in 0.5M dilute sulfuric acid solution for 10s, and 5V direct current is applied for electrolysis, then rinsed with a large amount of ultrapure water, then immersed in 0.5M dilute hydrochloric acid for 10s and repeatedly washed with ultrapure water, repeat the above steps twice, and the immersion time in the last dilute hydrochloric acid immersion should be at least 2min, and finally washed with ultrapure water and annealed with a butane gun.

[0035] The assembly process of the battery solution includes: the electrolytic cell used in the experiment is customized with polytetrafluoroethylene that is resistant to high temperature, acid and alkali corrosion and not easy to deform. In order to increase the fit between the electrolytic cell and the Au substrate, an O-ring is used to increase the sealing of the electrolytic cell. During the experiment, the substrate for fixing the Au (111) electrode is placed on the steel sheet, and then the electrolytic cell is installed and pressed, and finally fixed with screws to complete the installation.

[0036] The molecules in the solution that can be anchored with gold form a metal-molecule-metal junction with the needle tip. When the piezoelectric ceramic controls the needle tip to move away from the substrate, the metal-molecule-metal junction is broken, and the conductance-distance curve is recorded at the same time.

[0037] The experimental preparation process includes:

[0038] Put the electrolytic cell, O-rings, and several solvent bottles into a beaker, and soak them in a piranha solution with a volume ratio of concentrated sulfuric acid: 30% hydrogen peroxide = 3:1 for more than 1 hour. After soaking, pour the waste liquid into the waste liquid tank, and then wash the spare parts with a large amount of ultrapure water several times. Then put the beaker containing the spare parts on an electric stove and heat it until the ultrapure water boils 3 times. Finally, pour out the water in the beaker and put it in a 105℃ oven to dry for use.

[0039] Weigh 7.49 mg of NaI into a solvent bottle containing 10 mL of ultrapure water and dissolve it completely by ultrasonication to obtain 5 × 10 -3 mol / LNaI, take 1mL 5×10 -3 mol / LNaI is placed in a 10mL solvent bottle and 9mL of ultrapure water is added to obtain a concentration of 5×10 -4 mol / LNaI, repeat the above steps to prepare 5×10 -5-5×10 -9 0.71 mg Na2SO4, 0.70 mg NaClO4·H2O, 0.51 mg NaBr, and 0.29 mg NaCl were weighed and placed in a solvent bottle containing 10 mL ultrapure water to obtain 5×10 -4 mol / L interfering ion solution, gradually diluted to 5×10 -6 mol / L. Weigh 0.16 mg of 4,4'-bipyridine and place it in a 10 mL solvent bottle. Use a pipette to transfer 1 mL of a 5×10 -4 mol / LNaI, and then transfer 1mL of 1×10 -6 mol / L interfering ion solution, and then 8 mL of ultrapure water was added to obtain a 0.1 mM 4,4'-bipyridine aqueous solution containing 5×10 - 5 mol / L iodine ion and 5×10 -7 mol / L SO4 2- 、ClO4 - Br - , Cl- interfering ions. Weigh 0.88 mg ascorbic acid, 0.84 mg uric acid, 0.77 mg dopamine, and 0.60 mg cysteine ​​respectively and place them in a solvent bottle containing 10 mL ultrapure water to obtain 5×10 - 4 mol / L interfering molecule solution, gradually diluted to 5×10 -6 mol / L. Weigh 0.16 mg of 4,4'-bipyridine and place it in a 10 mL solvent bottle. Use a pipette to transfer 1 mL of a 5×10 -4 mol / LNaI, and then transfer 1mL of 1×10 -6 mol / L interfering molecule solution, and then 8 mL of ultrapure water was added to obtain a 0.1 mM 4,4'-bipyridine aqueous solution containing 5×10 -5 mol / L iodine ion and 5×10 -7 M ascorbic acid, uric acid, dopamine, and cysteine ​​interfering molecules. Weigh 0.16 mg of 4,4'-bipyridine and place it in a 10 mL solvent bottle. Then use a pipette to transfer 1 mL of a 5×10 -4 mol / LNaI, and then 9mL of artificial urine was added to obtain artificial urine with a concentration of 0.1mmol / L4,4'-bipyridine, which contained 5×10 -5 mol / L of iodide ions. Repeat the above operation to obtain 0.1mmol / L 4,4'-bipyridine containing iodide ions at concentrations of 5×10 -5 -5×10 -10 mol / L artificial urine.

[0040] Use a pipette to transfer about 200 μL of solution into the electrolytic cell, then cover it with a shield to reduce noise, and start testing when the entire device is stable. Set the STM bias to 100 mV, drive the needle tip to continuously approach the substrate and move away from the substrate at a speed of 20 nm / s after impact, and collect the current-distance curve at a sampling frequency of 20 KHz. Repeat the above steps until thousands of conductance-distance curves are collected.

[0041] The obtained conductivity-distance curve is plotted as a one-dimensional conductivity map, and the single-molecule conductivity peak area is obtained after Gaussian fitting of the conductivity peak in the one-dimensional conductivity map. The detection result of the iodide ion content in human urine is obtained based on the area of ​​the conductivity peak fitting.

[0042] According to the above steps, 5×10 -7 mol / L interfering ions (SO4 2- 、ClO4 - Br - , Cl-) and 5×10 -7 mol / L interfering molecules (ascorbic acid, uric acid, dopamine, cysteine), such as Figure 2 As shown in the figure, it can be seen that with the increase of iodine ion concentration, the conductivity peak area of ​​4,4'-bipyridine gradually weakens. The integration of its conductivity peak area can be linearly related to the negative logarithm of the iodine ion concentration. This strongly proves that the sensor we developed has high specificity for iodine ion sensing.

[0043] Next, the junction conductivity of 4,4'-bipyridine molecules with different iodide ion concentrations in artificial urine was measured, and the data collected in the experiment were log-statisticed and plotted into a one-dimensional conductivity histogram, as shown in Figure 1. Figure 3 As shown. With the increase of iodine ion concentration in artificial urine, the conductivity peak intensity of 4,4'-bipyridine weakens, and the conductivity peak intensity is proportional to the molecular binding rate. At the same time, the specific adsorption of iodine ions makes the conductivity peak area of ​​4,4'-bipyridine different in different iodine ion concentrations. Therefore, the conductivity peak area of ​​4,4'-bipyridine added to artificial urine with different iodine ion concentrations is plotted against the negative logarithm of the iodine ion concentration, and the following can be obtained: Figure 4 The working curve shown is Figure 4 By substituting the measured conductivity peak area into the working curve, the iodine content in urine can be obtained.

[0044] To further verify the working curve, 4,4'-bipyridine was added to simulated artificial urine with unknown iodine ion content to measure the conductivity. The experiment was repeated three times and the following results were obtained: Figure 5 The conductivity peak of 4,4'-bipyridine is shown in Figure 1. Substitute the conductivity peak area after Gaussian fitting into Figure 4 In the working curve, the iodine content in three experiments was obtained, and the average iodine content was calculated to be 225.2 μg / L. 3+ -Ce 4+ The average iodine content measured by catalytic spectrophotometry was 227.2 μg / L, and the difference between the two analysis methods was 0.9%, which confirmed the accuracy of the iodine ion detection method of the present invention.

[0045] The embodiments described above are only descriptions of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A single molecule electrical sensor for detecting the iodide ion content in human urine, characterized in that: The sensor comprises: a scanning tunneling microscope, a needle tip, a metal substrate, an electrolytic cell and an O-ring; the needle tip is inserted into the scanning head of the scanning tunneling microscope, the metal substrate is installed in the electrolytic cell, and the O-ring is arranged on the outside of the bottom of the electrolytic cell.

2. The single-molecule electrical sensor for detecting the iodine ion content in human urine according to claim 1, characterized in that: The metal substrate also includes gold balls, which are used to provide atomic-level flat gold single crystal electrodes for adsorbing the molecules to be detected.

3. A detection method of a single molecule electrical sensor for detecting the iodide ion content in human urine, the detection method using the single molecule electrical sensor according to any one of claims 1 to 2, characterized in that: The piezoelectric ceramic in the scanning tunneling microscope controls the needle tip to approach the metal substrate, collects the current value during the approaching process, and when the current value reaches a preset current value, the piezoelectric ceramic controls the needle tip to approach the substrate by a preset distance, and when the single-molecule electrical sensor detects the current value, the piezoelectric ceramic controls the needle tip to move away from the metal substrate at a constant speed; When the molecules in the electrolytic cell solution that have anchoring groups and can react with metals form a metal-molecule-metal junction between the needle tip and the metal substrate; When the needle tip is pulled upward, the connection between the molecule and the electrode is disconnected, completing a cleavage process; Collect the tunnel current during the splitting process to obtain the conductance-distance curve; The obtained conductivity-distance curve is plotted as a one-dimensional conductivity map, and the single-molecule conductivity value is obtained after Gaussian fitting of the conductivity peak in the one-dimensional conductivity map. The detection result of the iodide ion content in human urine is obtained based on the single-molecule conductivity peak area.

4. The detection method of the single molecule electrical sensor for detecting the iodide ion content in human urine according to claim 3, characterized in that: The preset range of the current value is 8nA to 80nA.

5. The detection method of the single molecule electrical sensor for detecting the iodide ion content in human urine according to claim 3, characterized in that: The needle tip is a gold wire with a length of 1.5 cm, a purity greater than or equal to 99.999%, and Φ=0.25 mm.

6. The detection method of the single molecule electrical sensor for detecting the iodide ion content in human urine according to claim 5, characterized in that: Use a 175° C. electric soldering iron to evenly apply hot melt adhesive to insulate and encapsulate the needle tip.

7. The detection method of the single molecule electrical sensor for detecting the iodine ion content in human urine according to claim 3, characterized in that: The metal substrate is a gold sheet with a thickness of 0.5 cm and a size of 10×15 mm.

8. The detection method of the single molecule electrical sensor for detecting the iodide ion content in human urine according to claim 3, characterized in that: The material of the electrolytic cell is polytetrafluoroethylene.

9. The detection method of the single molecule electrical sensor for detecting the iodide ion content in human urine according to claim 3, characterized in that: The process of obtaining the detection result of the iodine ion content in human urine based on the single molecule conductivity value specifically includes: 4,4'-bipyridine was added to urine for conductivity measurement, the conductivity peak in the conductivity measurement result was Gaussian fitted to obtain the peak area, the single molecule conductivity peak area was brought into the working curve to obtain the average iodine content in urine.

Images