KR20220135432A - Sandwich ELISA-Based Electrochemical Biosensor for leptin and leptin detecting method using the same - Google Patents

Abstract

The present invention relates to a leptin biosensor based on sandwich enzyme immunoassay and a leptin detecting method using the same, and more specifically, to technology for electrochemically detecting leptin by using an oxidation reaction of o-phenylenediamine (oPD) based on sandwich ELISA [DTSSP/leptin Ab (capture antibody)/leptin/biotinylated leptin Ab (detection antiboby)/streptavidin-HRP] in a screen-printed gold electrode (SPGE). When using the leptin biosensor based on sandwich enzyme immunoassay according to the present invention, a leptin concentration detecting curve is confirmed in a range of 0.1-20 ng/mL. Since the leptin biosensor provides high stability, sensitivity, selectivity, and effectiveness compared to commercial ELISA measurement, the biosensor is used at a medical institution and a point-of-care testing (POCT) level, and is used as a medical device to easily confirm scales of obesity and an obesity-mediated disease.

Description

Sandwich ELISA-Based Electrochemical Biosensor for leptin and leptin detecting method using the same}

The present invention relates to a sandwich enzyme immunoassay-based leptin biosensor and a leptin detection method using the same, and more particularly, sandwich ELISA [DTSSP/leptin Ab (capture antibody)/leptin/biotinylated leptin It relates to a technology for electrochemically detecting leptin using the oxidation reaction of o-phenylenediamine (oPD) based on Ab (detection antiboby)/streptavidin-HRP].

Leptin is a peptide hormone mainly produced in adipose tissue. Leptin secreted from fat is directly proportional to the blood concentration as the number and size of adipose tissue increases. Leptin and obesity have a close correlation, and in particular, obesity may accompany most obesity-mediated diseases, such as hypertension and cardiovascular disease.

In other words, leptin is considered to be an important biomarker for obesity and obesity-mediated diseases. Therefore, if the concentration of leptin in the blood can be measured using a biosensor capable of specifically detecting leptin, various physiological and pathological changes related to obesity in vivo can be easily identified. Therefore, measuring leptin levels is essential to provide medical information on obesity and obesity-mediated diseases.

On the other hand, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and Western blotting have been commonly used to measure leptin levels in most practical fields. Although these techniques are effective and well-established, they have limitations due to complexity, cost, and time-consuming procedures, in particular, require the use of large-scale equipment, skilled equipment technicians and complex protocols.

Electrochemical technology has advantages such as high sensitivity, adequate selectivity, low cost, time saving and long-term stability in detecting various types of molecules. In particular, electrochemical biosensors are attracting attention for measuring specific biomarkers (DNA, RNA, proteins, ions, and small molecules) in clinical and commercial fields. ELISA is one of the most effective and commonly used methods to quantify specific biomarkers with good performance.

Accordingly, there is a need to develop a new biosensor for efficient detection of leptin.

Korean Patent Registration No. 10-1053425 (July 27, 2011)

Therefore, an object of the present invention is based on a sandwich ELISA [DTSSP/leptin Ab (capture antibody)/leptin/biotinylated leptin Ab (detection antiboby)/streptavidin-HRP]) on a screen-printed gold electrode (SPGE) based on o-phenylenediamine An object of the present invention is to provide a biosensor for electrochemically detecting leptin using an oxidation reaction of (oPD) and a medical application thereof.

In order to achieve the above object, the present invention is a sandwich enzyme, characterized in that o-Penylenediamine (oPD) is applied as an electron mediator on a screen-printed gold electrode (SPGE) Provided are an immunoassay-based leptin biosensor and a method for manufacturing the same.

In addition, the present invention provides a kit for diagnosing obesity or obesity-mediated disease comprising the biosensor.

In addition, the present invention provides a method for detecting leptin, comprising treating a sample isolated from the human body with the biosensor.

In addition, the present invention provides a method for diagnosing obesity or obesity-mediated disease, comprising treating a sample isolated from the human body with the biosensor.

Using the sandwich enzyme immunoassay-based leptin biosensor according to the present invention, the leptin concentration detection curve was confirmed in the range of 0.1 to 20 ng/mL, and showed high stability, sensitivity, selectivity and effectiveness compared to commercial ELISA measurements. Therefore, the biosensor can be utilized as a medical device that can easily check the scale of obesity and obesity-mediated diseases by being utilized at the level of medical institutions and point-of-care testing (POCT).

1 is a schematic diagram (a) of a leptin biosensor based on a sandwich enzyme immunoassay according to the present invention, and a schematic diagram (b) of a diet-induced obesity (DIO) model development using C57BL/6J male mice.
2 is a comparison of the characteristics of high-fat diet (HFD) mice and normal diet (ND) mice, (a) is the appearance of ND and HFD mice, (b) is a graph showing the change in body weight over time in each experimental group, (c) is a graph showing the change in liver mass in each experimental group, (d) is a graph showing the change in epididymal mass in each experimental group, and (e) is a graph showing the change in subcutaneous fat mass in each experimental group.
3 shows the results of cyclic voltammetry (CV) electrochemical analysis of a leptin biosensor based on a sandwich enzyme immunoassay according to the present invention. (a) Cyclic voltammetry for stability at a scan rate of 10 to 300 mV/s (CV) analysis results, (b) oxidation and reduction strategy peaks from graph a, and (c) long-term storage capacity test results under a humidified condition of 4° C. over 18 days.
4 shows the results of square wave voltammetry (SWV) electrochemical analysis of a leptin biosensor based on a sandwich enzyme immunoassay according to the present invention, (a) for leptin solutions (0.1 to 20 ng/mL) of various concentrations. A square wave voltammetry (SWV) response curve of the biosensor, (b) a calibration plot showing a strong linear relationship between the SWV peaks is shown.

Hereinafter, the present invention will be described in more detail.

The present inventors made diligent efforts to develop a sandwich enzyme immunoassay-based leptin biosensor that exhibits high stability, sensitivity, selectivity and effectiveness compared to commercial ELISA measurements. As a result, a sandwich ELISA [DTSSP/leptin Based on Ab (capture antibody)/leptin/biotinylated leptin Ab (detection antiboby)/streptavidin-HRP], the present invention developed a technology for electrochemically detecting leptin using the oxidation reaction of o-phenylenediamine (oPD) was completed.

Accordingly, the present invention is a sandwich enzyme immunoassay-based leptin bioassay, characterized in that o-Penylenediamine (oPD) is applied as an electron mediator on a screen-printed gold electrode (SPGE). sensor is provided.

The biosensor comprises: a) a screen-printed gold electrode (SPGE); b) 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) formed on the electrode; c) an anti-leptin capture antibody that binds to said DTSSP and captures leptin; d) leptin; e) a biotin-bound anti-leptin detection antibody that detects the leptin; and f) streptavidin-horseradish peroxidase (HRP) bound to the anti-leptin detection antibody, and o-Penylenediamine (oPD) may be applied as an electron mediator.

The biosensor may diagnose obesity or obesity-mediated disease by detecting leptin, and the obesity-mediated disease may be one or more diseases selected from hypertension or cardiovascular disease, but is not limited thereto.

In addition, the present invention provides a kit for diagnosing obesity or obesity-mediated disease comprising the biosensor.

The obesity-mediated disease may be one or more diseases selected from hypertension or cardiovascular disease, but is not limited thereto.

In addition, the present invention provides a method for detecting leptin, comprising treating a sample isolated from the human body with the biosensor.

In addition, the present invention provides a method for diagnosing obesity or obesity-mediated disease, comprising treating a sample isolated from the human body with the biosensor.

The sample may be one or more selected from the group consisting of blood, plasma, urine, saliva, tissue, and cerebrospinal fluid, but is not limited thereto.

The obesity-mediated disease may be one or more diseases selected from hypertension or cardiovascular disease, but is not limited thereto.

In addition, the present invention comprises the steps of: a) forming 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) on a screen-printed gold electrode (SPGE); b) adding and reacting an anti-leptin capture antibody that binds to the DTSSP and captures leptin; c) adding an analyte material including leptin to the reacted reactant; d) detecting the leptin by adding an anti-leptin detection antibody to which biotin is bound; e) detecting said anti-leptin detection antibody by adding streptavidin-horseradish peroxidase (HRP); and f) adding and reacting o-phenylenediamine (oPD) as an HRP substrate.

The manufacturing method may further include washing a screen-printed gold electrode (SPGE) before step a).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Reference Example 1. Preparation of devices, electrodes and reagents

1.1. device and electrode

All electrochemical measurements were performed using a CHI760E electrochemical workstation purchased from CH Instruments (Austin, TX, USA). A screen-printed gold electrode [SPGE (DRP-C220AT)] was purchased from Dropsens. The gold electrode consisted of (i) a gold working electrode (diameter = 4 mm), (ii) a gold counter electrode and (iii) a silver reference electrode, and a cable connector connected the SPGE to the workstation.

1.2. reagent

All chemicals and reagents were analytical grade and used without further purification. Phosphate buffered saline (PBS), Triton X-100, o-phenylenediamine dihydrochloride (oPD), bovine serum albumin (BSA), potassium hexacyanoferrate III (K ₃ [Fe(CN) ₆ ]) and potassium Hexacyanoferrate II (K ₄ [Fe(CN) ₆ ]) trihydrate was purchased from Sigma-Aldrich (St. Louis. MO, USA). 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) was purchased from ThermoFisher. Streptavidin-HRP (horseradish peroxidase) solution was purchased from Abcam. The mouse leptin ELISA kit was purchased from Merck Millipore. Mouse leptin recombinant protein, mouse monoclonal anti-leptin antibody (capture antibody) and mouse polyclonal anti-leptin antibody (biotin-conjugated, detection antibody) were purchased from Enzo Life Sciences. All stock and buffer solutions were prepared using autoclaved Milli-Q ultrafiltration distillation and deionized water (18.2 MW- ^cm ). All experiments were performed at room temperature (23±1° C.).

Example 1. Diet-induced obesity mouse model

All animal testing procedures and protocols were approved by the Animal Care and Use Committee (KM-2019-18R2), Keimyung University School of Medicine. Male C57BL/6J mice (4 weeks old) were provided by Hyochang Science (Daegu, Korea), and were bred in standard cages at a temperature of 50%±5% relative humidity and 23±1° C. with a light-dark cycle for 12 hours.

Before changing the diet, the mice were acclimatized to the normal diet for one week, and all mice were divided into two weight-matched groups (n = 5 per group) into high-fat diet (HFD) mice and normal diet (ND) mice. . HFD mice were fed a high fat diet (60% kcal from fat), and the ND group was fed a normal diet (15% kcal from fat). Both groups were fed ad libitum for 12 weeks, and body weights were measured daily. After 15 hours of fasting, blood was collected by cardiac puncture using a K3 EDTA-coated tube and centrifuged at 4°C. Plasma was isolated and stored at 80°C until analysis. Organs and tissues (liver, epididymis and subcutaneous fat) were collected and then weighed.

1b shows a schematic diagram of DIO model development using male C57BL/6J mice, and in FIG. 2, the characteristics of high fat diet (HFD) mice and normal diet (ND) mice were compared. As shown in Figure 2b, the body weight of HFD mice (29.96 ± 3.33 g) was significantly increased than that of ND mice (21.33 ± 2.48 g). In addition, as shown in FIGS. 2c, 2d and 2e, the masses of liver, epididymis and subcutaneous fat of HDF mice were also significantly increased.

Thus, DIO mice were successfully established.

Example 2. Leptin biosensor fabrication (SPGE/DTSSP/anti-Leptin/Leptin/Biotinylated anti-leptin/streptavidin-HRP/oPD)

A schematic diagram of a sandwich ELISA-based electrochemical biosensor according to the present invention is shown in FIG. 1A.

First, before preparing the biosensor, SPGE was stored in pure ethanol. Impurities were removed at 4° C. overnight, and the mixture was gently dried under a flow of nitrogen gas. Then, 50 μL drops of 5 mM DTSSP solution were applied to the electrodes (work area only) and incubated for 2 hours. A 50 μL drop of anti-leptin antibody solution (10 μg/mL) was applied as capture antibody on the electrode for 1 hour. To prevent non-specific binding, blocking buffer (1% BSA, 1% Triton X-100) was placed on the electrode for 1 hour and then gently rinsed with blocking buffer. Then, 50 μL drops of antigen solution (leptin solution or mouse plasma) were applied onto the electrode and incubated at 4° C. in a humidified chamber. In addition, 50 μL drops of biotin-conjugated anti-leptin antibody solution (10 μg/mL) were applied on the electrode for 1 hour at room temperature. A streptavidin-HRP solution (0.1 μg/mL) was applied on the electrode at room temperature for 30 min.

To complete the fabrication of an ELISA-based electrochemical biosensor for leptin detection, a 50 μL droplet of oPD solution was placed as an electron mediator on the modified electrode, followed by incubation at room temperature for 30 min.

Example 3. Electrochemical detection

1. Electrochemical detection method

Two electrochemical detections were performed using the biosensor fabricated in Example 1; cyclic voltammetry (CV) and square-wave voltammetry (SWV).

During CV, cyclic voltammetry was recorded at potentials ranging from -0.5V to 0.7V with a scan rate of 100 mV/s. For SWV, a square wave voltammogram was recorded at a potential ranging from -0.8V to 0.0V with a frequency of 15 Hz and an amplitude of 0.025V. All experiments were performed and analyzed in triplicate at room temperature.

2. Stability

The stability and long-term storage capacity of the biosensor according to the present invention were analyzed. First, the stability of screen-printed gold electrodes (naked electrodes) without any modification by CV at various scan rates was evaluated. A 0.1M KCl solution containing 5 mM potassium ferricyanide (III) was applied at a scan rate of 10 to 300 mV/s. As shown in FIG. 3A , the absolute values of the cathode and anode peak currents showed a linear correlation with the square root of the scan rate. At this time, the correlation coefficients were 0.992 and 0.993 (Fig. 3b). These results confirm that the electrochemical reaction of the SPGE surface is a diffusion control process.

In addition, for analysis of the long-term storage capacity after storage of the biosensor according to the present invention, it was maintained under a blocking buffer (1% BSA containing 0.1% Triton X-100 in 1X PBS solution) at 4° C. for up to 18 days, followed by analysis did. As shown in FIG. 3c , it was confirmed that the electrochemical reaction of the biosensor after 18 days was maintained by 80.29% compared to the initial reaction measured on the first day.

3. Sensitivity

To analyze the sensitivity of the biosensor according to the present invention, quantitative detection of leptin was evaluated by SWV measurement for leptin solutions (0.1, 0.5, 1.0, 5.0, 10 and 20 ng/ml). The leptin solution was prepared with a mouse leptin recombinant protein having a specific binding affinity to a leptin antibody. Figure 4a shows the square wave voltammetry (SWV) response curve of the biosensor to various concentrations of leptin solution (0.1 ~ 20 ng/mL), and Figure 4b shows a strong linear relationship between the SWV peak current and the logarithm of the leptin concentration. A calibration plot (R ² =0.9867) is shown.

The limit of detection (LOD) of the biosensor according to the present invention was determined to be 0.033 ng/mL according to the 3σ rule, and the linearity and low LOD of the sandwich ELISA-based electrochemical biosensor according to the present invention are accurate and highly sensitive to mammalian physiology. This means that leptin concentrations can be quantified over a wide range of leptin levels.

4. Selectivity

The selectivity of the biosensor according to the present invention and the ability to identify specific biomarkers such as leptin in a mixture of body fluids such as plasma were examined. In Table 1 below, the leptin recovery rate was shown in a sample in which leptin, insulin, glucose and urea were mixed, and when the biosensor according to the present invention was used, the leptin recovery rate was nearly 100%.

[Table 1]

5. Efficiency

The efficacy of discriminating the leptin level in undiluted blood of two groups of biosensor-related, ND and HFD mice according to the present invention was examined. In Table 2 below, the leptin concentrations of the ND mouse group (3.08-5.86 ng/mL) and the HFD mouse group (11.58-18.58 ng/mL) using the biosensor according to the present invention are summarized.

In addition, as a result of comparing leptin levels using the biosensor according to the present invention and a microplate reader using a commercial ELISA kit, respectively, as shown in Table 2, the quantitative difference between the biosensor according to the present invention and the commercial ELISA kit was less than 10%. Confirmed.

[Table 2]

Summarizing these results, the biosensor according to the present invention exhibits high stability, sensitivity, selectivity and effectiveness compared to commercial ELISA measurement, and thus is utilized in medical institutions and POCT (point-of-care testing) levels, It can be used as a medical device that can easily check the scale of obesity-mediated diseases.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

A leptin biosensor based on sandwich enzyme immunoassay, characterized in that o-Penylenediamine (oPD) is applied as an electron mediator on a screen-printed gold electrode (SPGE). According to claim 1, wherein the biosensor,
a) screen-printed gold electrode (SPGE);
b) 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) formed on the electrode;
c) an anti-leptin capture antibody that binds to said DTSSP and captures leptin;
d) leptin;
e) a biotin-bound anti-leptin detection antibody that detects the leptin; and
f) comprising streptavidin-horseradish peroxidase (HRP) bound to the anti-leptin detection antibody, characterized in that o-Penylenediamine (oPD) is applied as an electron mediator, A leptin biosensor based on sandwich enzyme immunoassay. According to claim 1 or 2, wherein the biosensor,
A sandwich enzyme immunoassay-based leptin biosensor, characterized in that obesity or obesity-mediated disease is diagnosed through leptin detection. According to claim 3, wherein the obesity-mediated disease,
A leptin biosensor based on sandwich enzyme immunoassay, characterized in that at least one disease selected from hypertension or cardiovascular disease. A kit for diagnosing obesity or obesity-mediated disease comprising the biosensor according to claim 1 or 2. According to claim 5, wherein the obesity-mediated disease,
A kit, characterized in that at least one disease selected from hypertension or cardiovascular disease. A method for detecting leptin, comprising treating a sample isolated from a human body with the biosensor according to claim 1 or 2. The method according to claim 7, wherein the sample is at least one selected from the group consisting of blood, plasma, urine, saliva, tissue, and cerebrospinal fluid. A method for diagnosing obesity or obesity-mediated disease, comprising treating a sample isolated from a human body with the biosensor according to claim 1 or 2. 10. The method of claim 9, wherein the obesity-mediated disease,
A diagnostic method, characterized in that at least one disease selected from hypertension or cardiovascular disease. a) forming 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) on a screen-printed gold electrode (SPGE);
b) adding and reacting an anti-leptin capture antibody that binds to the DTSSP and captures leptin;
c) adding an analyte material including leptin to the reacted reactant;
d) detecting the leptin by adding an anti-leptin detection antibody to which biotin is bound;
e) detecting said anti-leptin detection antibody by adding streptavidin-horseradish peroxidase (HRP); and
f) A method for preparing a leptin biosensor based on a sandwich enzyme immunoassay, comprising the step of reacting by adding o-Penylenediamine (oPD) as an HRP substrate. 12. The method of claim 11, further comprising cleaning a screen-printed gold electrode (SPGE) prior to step a).

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