Rapid salivary pepsin test

Abstract

Pepsin can be found in saliva in patients with heartburn, particularly during postprandial periods. Pepsin in saliva, in concentration more than 200 ng / mL is related to stomach problem. The invention relates to a pepsin lateral flow device (LFD) and method for detecting salivary pepsin as a surrogate marker for gastroesophageal reflux disease (GERD). This method is simple, low cost and sensitive for rapid determination of pepsin.

Description

[0001] RAPID SALIVARY PEPSIN TEST

[0002] Technical Field

[0003] The invention relates to a pepsin lateral flow device (LFD) and method for detecting salivary pepsin as a surrogate marker for gastroesophageal reflux disease (GERD).

[0004] State of the Art

[0005] Biotechnology rapid diagnostic test (RDT) is a quick and easy-to-implement medical diagnostic test. RDTs are suitable for preliminary or emergency medical screening and for use in medical facilities with limited resources. The European Union defines rapid testing as medical devices capable of qualitative or semi quantitative in vitro diagnostics, used alone or in small batches, containing non-automatic procedures and designed to give rapid results. Lateral flow tests are probably the most well-known rapid diagnostic tests, similar to pregnancy tests, but other systems are available, such as measuring rods, vertical flow, etc.

[0006] Since the kits are based on methods such as real-time polymerase chain reactions, IgM ELISA tests, and microneutralization tests, samples are taken from the nose, sputum test, blood sample, throat swab, and nasal aspiration for testing. Demand for the sector is increasing as a result of the global coronavirus outbreak. While the number of patients in hospitals is increasing rapidly, the need for test kits to diagnose patients with symptoms is also increasing and leading to the growth of the market.

[0007] The kits of the prior art are troublesome as they require short-lived and multitasking processes. In the prior art, complex, expensive, and expertise-requiring methods such as fluorimetric and / or spectrophotometric analyses are used for pepsin analysis.

[0008] Brief Description of the Invention

[0009] Rapid and inexpensive diagnosis of disease markers or biochemical factors is one of the current challenges in medical science. With the invention, a pepsin kit that can perform simple, costefficient and rapid analysis for the semi quantitative measurement of amino acids is produced using the capabilities of molecularly suppressed polymers and solid phase extraction.

[0010] Detailed Description of the Invention The pepsin lateral flow device (LFD) of the invention, and the method used by this device, is a rapid, non-invasive test for detecting salivary pepsin as a surrogate marker for gastroesophageal reflux disease (GERD). Test sensitivity, specificity, and positive and negative predictive values (PPV, NPV) were obtained in patients with symptomatic and objective GERD evidence compared to healthy controls.

[0011] Coating of pepsin printed polymer on the surface of TiO2 / Chitosan nanoparticles

[0012] Pepsin (0.5 mmol) and methacrylic acid (MAA, 4 mmol or 0.34 mL) were dissolved in 25 mL of acetonitrile and stored for 12 hours before polymerization. Using ultrasonic vibration, the pre-synthesized 0.2 g TiO2 / chitosan (TiCh enriched chitosan) nanoparticles were dispersed in an ethanol solution (25 mL). Ethylene glycol dimethacrylate (EGDMA, 20 mmol or 3.78 mL) and 2,2'-azobisisobutyronitrile (AIBN, 2 mmol or 0.332 g) from the pre-arranged solution were then dissolved in said solution. The combined solution was cleaned with high-purity argon and cooled in an ice bath for 15 minutes. At a speed of 300 RPM, the polymerization reaction was carried out at a three-stage temperature. Pre-polymerization was performed at 50°C for 6 hours, followed by polymerization at 60°C for 24 hours. The products were then polymerized for another 6 hours at 75°C to achieve a high cross-linking density. The temperature was increased from 60°C to 75°C at a rate of 0.25°C / min within 1 hour. Centrifugation was used to separate the resulting polymer from the combined solution. The unprinted polymer was synthesized in the same manner as described above but without the use of a template. The nanoparticles obtained were then used in the Soxhlet extractor in the presence of acetonitrile to create the template, then washed with methanol and dried under vacuum at room temperature.

[0013] Preparation of PVDF-impregnated pepsin-printed polymer

[0014] First of all, 100 mg of polyvinylidene fluoride (PVDF) was dissolved in dimethyl sulfoxide (DMSO) solvent, 1 mL of hydrogen peroxide (H2O2) was added to increase its hydrophilic property, and 30 mg of pepsin-specific molecular printed polymer was added and homogenized in an ultrasonic bath. This mixture was then placed in the Eppendorf tube and dried in a vacuum oven at 90°C for 5 hours, and the PVDF-impregnated pepsin-printed polymer was ready.

Claims

CLAIMS1. A lateral flow device for determining the level of pepsin in the stomach, characterized in comprising pepsin printed polymer coated on TiCh enriched nanoparticles.

2. The device according to Claim 1, characterized in that the polymer is polymethacrylic acid.

3. Manufacturing method of pepsin-printed polymer coated on TiO2-enriched nanoparticles, characterized in comprising steps of a. Keeping pepsin and methacrylic acid away from light for 12 hours before polymerization after dissolving in acetonitrile b. Dispersing previously synthesized TiO2-enriched chitosan nanoparticles in an ethanol solution using ultrasonic vibration c. Adding ethylene glycol dimethacrylate and 2,2'-azobisisobutyronitrile d. Cooling the obtained solution by cleaning with high-purity argon e. Performing pre-polymerization at 50°C for 6 hours f. Performing polymerization at 60°C for 24 hours g. Performing polymerization at 75°C for another 6 hours to achieve a high crosslinking density h. Dissolving polyvinylidene fluoride in dimethyl sulfoxide in a separate place, adding hydrogen peroxide to increase its hydrophilic property i. Adding pepsin-printed polymer to poly vinylidene fluoride solution j. Drying