A portable dual-channel sensor for rosiglitazone content detection

Abstract

The invention discloses a portable dual-channel sensor for detecting the content of rosiglitazone. The cathode of the dual-channel sensor is a Prussian blue film, and the anode of the dual-channel sensor is a Cu 2 O@Au loaded with secondary antibody modified glassy carbon electrode. 2 The excellent catalytic activity of O@Au is combined with the timing detection to realize the change of time signal in the immune detection. At the same time, the electrochromic performance of the PB film is also time-dependent, thereby realizing dual-channel detection, making the sensing method more reliable and accurate. In addition, the present invention can realize real-time data reading, and can detect the content of rosiglitazone more quickly and conveniently. It has the advantages of low cost, dual-channel readout, simple operation, etc., and provides a new method for portable and rapid detection.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection and analysis instruments, and in particular relates to a portable dual-channel sensor for detecting the content of rosiglitazone. Background Art

[0002] There are a large number of diabetic patients in China, so the demand for hypoglycemic drugs and health products is very huge. Driven by profit, some unscrupulous merchants add various additives to these drugs or products to achieve the effect of rapidly lowering blood sugar. Among these additives, rosiglitazone (RSG) is widely used in the treatment of type II diabetes because it can better promote the human body's use of insulin. However, excessive intake of RSG will increase the risk of stroke, heart attack, heart failure, myocardial infarction and even cardiovascular death. Therefore, it is of great significance to establish an effective detection method for RSG in hypoglycemic drugs and health products.

[0003] Conventional RSG detection methods include liquid chromatography-mass spectrometry, ultraviolet-visible spectroscopy, Raman spectroscopy, etc. These methods provide sensitive and reliable methods for RSG detection, but they also have obvious shortcomings. For example, the operation cost is high, it is time-consuming, and professional analytical instruments and operators are required, which to a certain extent limits the promotion of these methods, especially in areas with scarce resources, where the application of these technologies is more difficult. Summary of the invention

[0004] The object of the present invention is to overcome at least one disadvantage of the prior art and provide a dual-channel sensor for detecting the content of rosiglitazone.

[0005] The technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a portable dual-channel sensor for detecting rosiglitazone content, wherein the cathode of the dual-channel sensor is a Prussian blue film, and the anode of the dual-channel sensor is a glassy carbon electrode modified with a Cu2O@Au loaded secondary antibody.

[0007] In some examples, the cathode of the dual-channel sensor is prepared by loading Prussian blue on the surface of FTO conductive glass using an electrochemical deposition method.

[0008] In some examples, the electrochemical deposition current is 5 to 50 μA, and the electrochemical deposition time is 10 to 100 seconds.

[0009] In some examples, the method for preparing the anode of the dual-channel sensor includes the following steps:

[0010] 1) Synthesis of Cu2O@Au loaded secondary antibody: First, Cu2O nanoparticles were synthesized, then mixed with auCl, then modified with thiourea, and finally added with goat anti-rabbit secondary antibody to obtain Cu2O@Au loaded secondary antibody;

[0011] 2) Preparation of electrochemical immunosensor: First, the chitosan@multi-walled carbon nanotubes@polydopamine composite material was dropped onto the surface of the glassy carbon electrode, and then the glassy carbon electrode was modified with rosiglitazone antigen and blocking agent. Subsequently, rosiglitazone antibody was added and incubated with rosiglitazone. Finally, a mixed solution containing 4-chloro-1-naphthol and H2O2 was added for reaction, washing and drying to obtain a glassy carbon electrode modified with Cu2O@Au loaded secondary antibody.

[0012] In some examples, the preparation method of Cu2O nanoparticles is: adding CuCl2·2H2O to a polyvinyl pyrrolidone aqueous solution and stirring, then adding an inorganic base solution and mixing evenly, then adding an ascorbic acid solution for reduction reaction, centrifuging, washing, and drying to obtain Cu2O nanoparticles.

[0013] In some examples, the preparation method of the chitosan@multi-walled carbon nanotubes@polydopamine composite material is: dissolve chitosan and then add multi-walled carbon nanotubes, stir evenly, and then add the mixed solution and dopamine hydrochloride to a buffer solution in sequence and mix evenly to obtain a chitosan@multi-walled carbon nanotubes@polydopamine composite material.

[0014] In some examples, the volume ratio of the rosiglitazone antibody to rosiglitazone is (0.9-1.1):1.

[0015] In some examples, the incubation time is 0.1-5.0 h, and the incubation temperature is 35-40° C.

[0016] In a second aspect, the present invention provides use of the portable dual-channel sensor described in the first aspect in the detection of rosiglitazone, comprising the following steps:

[0017] 1) First, the glassy carbon electrode is modified with antigen and blocking agent, and then rosiglitazone standard solutions with a certain concentration gradient are prepared respectively, and then the standard solutions are used to prepare the Cu2O@Au loaded secondary antibody modified glassy carbon electrode;

[0018] 2) Then, the glassy carbon electrode obtained in step 1 is used as an anode, and the cathode is a Prussian blue film, which is placed in an electrolytic cell and energized, and then the time and the colorimetric information of the Prussian blue film are recorded, so as to construct standard curves between rosiglitazone concentration and time, and between rosiglitazone concentration and the colorimetric information of the Prussian blue film;

[0019] 3) preparing a rosiglitazone solution to be tested, performing the test according to the method of step 1) and step 2), and finally calculating the rosiglitazone concentration in the sample to be tested according to the standard curve of step 2.

[0020] In a third aspect, the present invention provides a portable rosiglitazone content detection device, comprising an electrolyte, a time processor, an image processor and a dual-mode sensor, wherein the dual-mode sensor is the dual-channel sensor described in the first aspect.

[0021] The beneficial effects of the present invention are as follows: the present invention develops a dual-channel sensor for the "timing-colorimetry" dual-channel detection of rosiglitazone. The excellent catalytic activity of Cu2O@Au is combined with timing detection to achieve the change of the time signal in the immunoassay. In addition, the electrochromic performance of the PB film also shows time dependence, thereby realizing dual-channel detection, making the sensing method more reliable and accurate. Moreover, the results of the two signal channels are independent of each other and can be verified with each other, which improves the reliability and sensitivity of the results. Moreover, the present invention records the timing signal based on the time readout device and converts it into a rosiglitazone concentration signal, and transmits the signal to the mobile phone APP (RSG Sensor), so that the data can be read in real time. Therefore, the present invention has the advantages of low cost, dual-channel readout, simple operation, etc., and provides a new method for portable and rapid detection of illegal additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The structural details of the TBR device;

[0023] Figure 2 Characterization diagram of Cu2O@Au nanomaterials;

[0024] Figure 3 Figure 2 is a performance graph of a dual-channel sensor based on a TBR device. DETAILED DESCRIPTION

[0025] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention.

[0026] Example

[0027] 1) Synthesis of Cu2O@Au loaded secondary antibody (Cu2O@Au-Ab2):

[0028] First, Cu2O nanoparticles with octahedral structure are synthesized. CuCl2·2H2O (0.1-1.0 g) is added to a polyvinylpyrrolidone (PVP) aqueous solution (0.1-1 wt.%), 10 mL of NaOH (1-10 M) is added, and the mixture is stirred at 30-60°C for 10-60 minutes, and then 1-10 mL of ascorbic acid (0.1-1.0 M) is added, and the mixture is reacted at 55°C for 1.5 hours. The mixture is centrifuged at 4000-10000 rpm for 5-20 minutes, the product is collected, washed with water and alcohol by centrifugation 3 times each, dried under vacuum, and the obtained powder is dispersed in 10-50 mL of ethanol.

[0029] Secondly, Cu2O@Au-Ab2 was prepared. 0.1-0.5 mL of Cu2O solution was dispersed in 0.1-1.0 mL of H2O and then mixed with 0.5-5.0 mL of HAuCl4. After vigorous shaking for 30-100 seconds, the product was collected by centrifugation, washed with ethanol, dried and dispersed in 0.5-5.0 mL of water. In order to modify the amino group, 0.5-5.0 mL of Cu2O@Au solution was mixed with 1 mL of thiourea (0.1-5.0 μM) and stirred at 37°C overnight. The amino-modified Cu2O@Au was washed, collected by centrifugation, dispersed in 0.1-5.0 mL of PBS (pH 7.4), and 5-20 μL of secondary antibody (Ab2) was added and stirred at 4°C for 24 hours. After washing with PBS, the final product Cu2O@Au-Ab2 was dispersed in 1 mL of PBS.

[0030] 2) Preparation of electrochemical immunosensor:

[0031] First, chitosan@multi-walled carbon nanotubes@polydopamine (CS@MWCNT@PDA) composite materials were prepared. The preparation process is as follows: 0.1-5.0g chitosan (CS) was dissolved in a 2% acetic acid solution, 10-50mg multi-walled carbon nanotubes (MWCNT) were added, and the mixture was stirred evenly and ultrasonicated for 1 hour, which was recorded as CS@MWCNT. Then 50-500μLCS@MWCNT solution and 0.5-5.0mg dopamine hydrochloride were added to Tris-HAc buffer (pH 8.5), and ultrasonicated for 1-50 minutes to obtain CS@MWCNT@PDA. Secondly, an immune reaction was carried out on the electrode surface. Specifically, 0.1-5.0μLCS@MWCNT@PDA was first added dropwise to the surface of the glassy carbon electrode (GCE), and after drying, CS@MWCNT@PDA could form a uniform film on the surface of the GCE. Then, 0.1-5.0 μL of antigen was dropped on the GCE and incubated overnight at 4°C, and 0.1-5.0 μL of blocking agent was added to cover the remaining active sites. In the immunocompetitive assay, 0.1-5.0 μL of a solution containing rosiglitazone (RSG) and antibody (V:V=1:1) was dropped on the GCE and incubated at 37°C, and then 0.1-5.0 μL of Cu2O@Au-Ab2 was dropped on the GCE and incubated at 37°C for 0.1-5.0 h. In each step, the electrode surface was washed with phosphate buffered saline (PBS). Finally, 0.1-5.0 μL of a mixed solution containing 4-chloro-1-naphthol (4-CN) and 10-200 mM H2O2 was added and reacted for 30 minutes. After washing and drying, the GCE was used for electrochemical testing.

[0032] 3) Preparation of Prussian blue (PB) film:

[0033] PB was loaded on the surface of FTO conductive glass by electrochemical deposition. First, 0.1-5.0g K3[Fe(CN)6] and 0.1-5.0g FeCl3·6H2O were dissolved in 5-50mL water, and 10mL HCl (0.01-0.5M) was added and stirred evenly to prepare the electrodeposition precursor solution. Electrodeposition was carried out by chronopotentiometry, in which the working electrode, counter electrode, and reference electrode were FTO, platinum electrode, and Ag / AgCl electrode, respectively. The cathode current was set to 5-50μA, and the electrodeposition time was set to 10-100 seconds.

[0034] 4) Development of portable time-based readout (TBR) devices:

[0035] The device uses a dual-electrode system, in which GCE is the bioanode and PB film is the cathode. In addition, an APP called "RSG Sensor" was developed to receive RSG concentration information. When using it, first connect the APP to the Bluetooth module of the device by clicking the "ConnectBluetooth" button at the bottom; then immerse the two electrodes in a 1M KCl solution to trigger the device's automatic start timing program. When the potential reaches the cut-off potential (E cut ), the timing program stops, the device will record the corresponding time, convert the time information into RSG concentration, and transmit it to the APP synchronously. After each test, click the "Clear" button to clear the reading.

[0036] The specific circuit design of the device is as follows Figure 1 As shown in Figure A, a DC battery (i) is used as a power supply, a fixed resistor (ii) is used to adjust the circuit potential, and an integrated circuit (IC) (iii) is connected in parallel with a dual-electrode system (iv) as the core component of a smart device. The IC has the function of monitoring potential and timing, and its details are as follows: Figure 1 B, where a and b are respectively the Bluetooth module (a), power interface (b), power switch (c), core chip (d), crystal oscillator (e) and two-electrode system interface (f). The core chip is used to read the real-time potential value, the crystal oscillator is used for high-precision timing, and the Bluetooth module is used to connect to the mobile phone APP "RSGSensor" ( Figure 1 C) The actual picture of the device is as follows Figure 1 As shown in D, 1-3 in the figure are IC, lithium battery and electrolytic cell respectively.

[0037] During the detection, the consumption of Cl- on the surface of the glassy carbon electrode can cause the partial voltage (E) to continue to increase. When E increases to the specified cut-off potential (E cut ), the timing program can stop automatically and record the time when E cutThe time required (t) is sent to the mobile phone APP "RSG Sensor" via Bluetooth and converted into RSG concentration information by the APP. E should be considered in the process of building the device. cut Since the time dependency between E and t only holds when t is less than the transition time (τ), we can achieve E cut The time required should be less than or equal to the corresponding τ. cut , GCE was modified with antigen, antibody, Cu2O@Au-Ab2, and a mixed solution of 4-CN and 100mM H2O2 in sequence, and then placed in the TBR device for timing detection. At this time, the multimeter measured the initial current in the circuit to be 4.8μA. Under this condition, E( Figure 1 E). As can be seen from the figure, E (black solid line) shows an upward trend with time, and the derivative curve of E to t (red dotted line) reaches a peak at 187 seconds, indicating that the transition time (τ) is 187 seconds; the corresponding E at this time is 1.15V, so 1.15V is set as E cut In addition, the figure is basically consistent with the chronopotentiometry curve obtained from the electrochemical workstation ( Figure 1 E), indicating that the device has a certain degree of reliability.

[0038] 5) Characterization of Cu2O@Au

[0039] Due to Cu 2+ / Cu2O has a lower potential (0.20V vs SHE) than AuCl4 - / Au potential (0.99V vs SHE), so AuCl4 - Cu2O can be oxidized, and Au can nucleate and grow on the surface of Cu2O. The SEM image of Cu2O@Au is shown in Figure 2 As shown in A, Cu2O has an octahedral structure and AuNPs are evenly distributed on the surface of Cu2O ( Figure 2 A inset). From the X-ray energy spectrum (EDS, Figure 2 B) shows the presence of Cu, O and Au, and the element distribution diagram ( Figure 2 C) shows that these elements are evenly distributed. In addition, Cu, Au, and O elements can be found in the full X-ray photoelectron spectroscopy (XPS) spectrum of Cu2O@Au ( Figure 2 D). The above characterizations demonstrate the successful synthesis of Cu2O@Au nanomaterials.

[0040] 6) Experimental method for detecting RSG samples

[0041] (1) Coating: 2 μL of CS@MWCNT@PDA was dropped onto the surface of glassy carbon electrode (GCE). After drying, CS@MWCNT@PDA formed a uniform film on the surface of GCE. Then, 2 μL of antigen was dropped onto GCE and incubated overnight at 4°C.

[0042] (2) Washing: Wash the GCE twice with PBS, 250 μL each time, and blow dry with N2.

[0043] (3) Blocking: 2 μL of 5% bovine serum albumin was added to the GCE surface and incubated at 37°C for 12 h to block the excess sites.

[0044] (4) Washing: Wash the GCE modified with BSA twice with PBS, 250 μL each time, and blow dry with N2.

[0045] (5) Preparation of standard samples: First, prepare the samples with a concentration of 5×10 -10 g / L, 1×10 -9 g / L, 5×10 -9 g / L, 1×10 -8 g / L, 5×10 -8 g / L, 1×10 -7 g / L and 5×10 -7 g / L RSG solution, and then mix the antibody and the above solutions of different concentrations at a volume ratio of 1:1. Add 2 μL of the mixed solution dropwise onto the GCE surface and incubate at 37°C for 2 h.

[0046] (6) Washing: Wash the GCE modified with the antibody and RSG twice with phosphate-buffered saline + Tween (PBST) buffer, 250 μL each time, and blow dry with N2.

[0047] (7) Simulated enzyme-labeled secondary antibody: 2 μL of Cu2O@Au@Ab2 was dropped on GCE and incubated at 37°C for 2 h. After washing twice with PBS and drying with N2, 2 μL of a mixed solution containing 4-CN and 100 mM H2O2 (v:v=1:1) was added and reacted for 30 min. After the reaction was completed, the solution was washed twice with PBS and dried with N2.

[0048] (8) Drawing of standard curve: The modified GCE was used as the working electrode and the platinum electrode was used as the counter electrode to connect to the TBR device and place it in an electrolytic cell containing 1M KCl solution. The TBR device was powered on and timed. When the system voltage rose to the specified voltage, the TBR device was automatically powered off, and the mobile phone recorded the time (t) of the system power on. At the same time, the colorimetric information (B / (R+G+B)) of the Prussian blue flakes could be directly obtained through the image processing software, thereby constructing a standard curve between RSG concentration and t and (B / (R+G+B)).

[0049] (9) Determination of unknown samples: Test the sample according to steps 5 to 7 and compare it with the standard curve to obtain the RSG concentration in the sample.

[0050] 7) Dual-channel sensor performance test based on TBR equipment

[0051] Since Cu2O@Au has the activity of peroxidase mimicking enzyme, 4-CN can be catalyzed to generate insoluble precipitate (B4Q) in the presence of Cu2O@Au / H2O2. The generation of B4Q increases the resistance, reduces the electron transfer rate constant, reduces the circuit current, and reduces the growth rate of the system partial pressure (E). Therefore, at different concentrations of RSG, the time (t) required for E to reach a specific value is different. In the timing channel, with the increase of RSG concentration, t shows a downward trend ( Figure 3 A). Figure 3 As shown in B, the square root of t The correlation equation with the logarithm of RSG concentration is: (R 2 =0.983)(C: RSG concentration), and its linear range is 5×10 -10 -5×10 -7 g / L, the detection limit (LOD) was 8.2×10 -11 g / L(S / N=3).

[0052] On the other hand, Prussian blue (PB) film was used as the cathode of the device for visual electrochromic detection. Under electrical stimulation, PB can show a clear change from blue to white, and this color change is rapid and reversible. The blue depth of PB film increases with the increase of RSG concentration ( Figure 3 C inset), at 5×10 -10 -5×10 -7 g / L, the color value (B / (R+G+B)) is linearly correlated with the logarithm of RSG concentration ( Figure 3 C), the equation is (B / (R+G+B))=0.02lgC+0.55(R 2 =0.985), LOD is 1.3×10 -10 g / L(S / N=3).

[0053] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A portable dual-channel sensor for detecting rosiglitazone content, characterized in that: The cathode of the dual-channel sensor is a Prussian blue film, and the anode of the dual-channel sensor is a glassy carbon electrode modified with Cu2O@Au loaded secondary antibody; the preparation method of the anode of the dual-channel sensor includes the following steps: 1) Synthesis of Cu2O@Au loaded secondary antibody: First, Cu2O nanoparticles were synthesized, then mixed with auCl, then modified with thiourea, and finally added with goat anti-rabbit secondary antibody to obtain Cu2O@Au loaded secondary antibody; 2) Preparation of electrochemical immunosensor: First, chitosan@multi-walled carbon nanotubes@polydopamine composite material was dropped onto the surface of glassy carbon electrode, and then the glassy carbon electrode was modified with rosiglitazone antigen and blocking agent, followed by adding rosiglitazone antibody and incubating with rosiglitazone, and adding the Cu2O@Au loaded secondary antibody prepared in step 1) on the glassy carbon electrode and incubating at 35-40 °C for 0.1-5.0 h, and finally adding a mixed solution containing 4-chloro-1-naphthol and H2O2 for reaction, washing and drying to obtain a glassy carbon electrode modified with Cu2O@Au loaded secondary antibody.

2. The portable dual-channel sensor according to claim 1, characterized in that: The cathode of the dual-channel sensor is prepared by loading Prussian blue on the surface of FTO conductive glass using an electrochemical deposition method.

3. The portable dual-channel sensor according to claim 2, characterized in that: The electrochemical deposition current is 5 to 50 μA, and the electrochemical deposition time is 10 to 100 seconds.

4. The portable dual-channel sensor according to claim 1, characterized in that: The preparation method of the Cu2O nanoparticles in step 1) is as follows: adding CuCl2×2H2O to a polyvinyl pyrrolidone aqueous solution and stirring, then adding an inorganic alkali solution and mixing evenly, then adding an ascorbic acid solution for reduction reaction, centrifuging, washing, and drying to obtain Cu2O nanoparticles.

5. The portable dual-channel sensor according to claim 1, characterized in that: The preparation method of the chitosan@multi-walled carbon nanotubes@polydopamine composite material in step 2) is: dissolving chitosan and then adding multi-walled carbon nanotubes, stirring evenly, and then sequentially adding the mixed solution and dopamine hydrochloride into a buffer solution and mixing evenly to obtain the chitosan@multi-walled carbon nanotubes@polydopamine composite material.

6. The portable dual-channel sensor according to claim 1, characterized in that: The volume ratio of rosiglitazone antibody to rosiglitazone added in step 2) is (0.9-1.1):

1.

7. Use of the portable dual-channel sensor according to any one of claims 1 to 6 in the detection of rosiglitazone, characterized in that: The following steps are involved: First, the glassy carbon electrode was modified with antigen and blocking agent, and then rosiglitazone standard solutions with a certain concentration gradient were prepared respectively, and then the standard solutions were used to prepare the Cu2O@Au loaded secondary antibody modified glassy carbon electrode; Then, the glassy carbon electrode obtained in step 1 is used as an anode, and the cathode is a Prussian blue film, which is placed in an electrolytic cell and energized, and then the time and the colorimetric information of the Prussian blue film are recorded, so as to construct standard curves between rosiglitazone concentration and time, and between rosiglitazone concentration and the colorimetric information of the Prussian blue film, respectively; Prepare the rosiglitazone solution to be tested, perform the test according to the method of step 1) and step 2), and finally calculate the rosiglitazone concentration in the sample to be tested according to the standard curve of step 2.

8. A portable rosiglitazone content detection device, comprising an electrolyte, a time processor, an image processor and a dual-mode sensor, characterized in that: The dual-mode sensor is a portable dual-channel sensor as claimed in any one of claims 1 to 6.

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