WO2024063638A1 - Device for detecting albumin from saliva - Google Patents

Device for detecting albumin from saliva Download PDF

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Publication number
WO2024063638A1
WO2024063638A1 PCT/MY2023/050086 MY2023050086W WO2024063638A1 WO 2024063638 A1 WO2024063638 A1 WO 2024063638A1 MY 2023050086 W MY2023050086 W MY 2023050086W WO 2024063638 A1 WO2024063638 A1 WO 2024063638A1
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Prior art keywords
albumin
electrode
imprinted polymer
molecularly imprinted
pyrrole
Prior art date
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PCT/MY2023/050086
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French (fr)
Inventor
Muhamad MAT SALLEH
Tengku Hasnan TENGKU ABDUL AZIZ
Ahmad Rifqi MD ZAIN
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Universiti Kebangsaan Malaysia
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Publication of WO2024063638A1 publication Critical patent/WO2024063638A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes

Definitions

  • the present invention relates to a medical device for detecting protein, particularly a medical device based on electrochemical methods for detecting albumin from human saliva.
  • Albumin is a type of protein found in blood. Liver makes albumin. An albumin blood test is used to check the functions of liver and kidney. Albumin has a role in repairing tissue and helping the body grow while transporting vital hormones and nutrients around.
  • Abnormal albumin level could indicate a number of conditions such as liver disease, cirrhosis, kidney disease, malnutrition, infection, inflammatory bowel disease and thyroid disease.
  • a typical albumin blood test involves a thin needle to take blood from the vein. The blood is then filled in a tube. The tube is then sent to a lab for analysis. A protein electrophoresis machine is used to indicate the albumin level. It is a common procedure when a person undergoes a medical body check-up.
  • Henskens investigated salivary protein, albumin and cystatin concentrations. Shaila shown that salivary albumin was assessed using the Bromoscresol green method. The reaction between albumin in saliva and dye Bromoscresol green produces change in colour which is proportional to albumin concentration in saliva. The colour is estimated using a photoelectric colorimeter at wavelength of 630 nm.
  • US2010/0167306 disclosed a rapid test for glycated albumin in saliva.
  • Saliva albumin is derived from plasma albumin and therefore contains glycated and non-glycated albumin fractions. The ratio of glycated albumin to total albumin in saliva is measured.
  • the test kit is placed in a spectrophotometer or fluorometer to determine the ratio.
  • the albumin blood test is an invasive procedure that involves the drawing of blood. The procedure is time consuming as it needs to be sent to a lab with protein electrophoresis machine. Other described method also describes the use of machine to detect albumin. A simpler rapid test for detecting albumin without the use of machine is desired.
  • the present invention discloses a device for detecting albumin from saliva.
  • a screen-printed electrode is modified to detect albumin.
  • the screen-printed electrode has a counter electrode, a working electrode and a reference electrode.
  • the working electrode is modified with a molecularly imprinted polymer (MIP) based on albumin analyte and pyrrole.
  • MIP molecularly imprinted polymer
  • the MIP is derived from pyrrole, perchlorate salt and Bovine Serum Albumin. A drop of MIP was made on the working electrode. The area of MIP is 40 to 60 ⁇ m 2 .
  • the screen-printed electrode is made in contact with saliva. Then, it is inserted in an Ampere reader to determine the level of albumin
  • FIG. 1 is a diagram of a modified surface-printed electrode according to the invention.
  • Electrochemical sensors are gaining traction in instrumental analysis. Screen printing is a method to produce electrochemical sensors. This technology is used to prepare single use screen-printed electrodes (SPEs). SPEs surface can be functionalised to enhance electrochemical reactivity and sensitivity for specific analytes.
  • SPEs are attractive because it uses a small volume of samples. SPEs are also convenient as it is a form of in-situ test.
  • Molecular imprinting uses target as a template during polymerization of functional monomers. Subsequent removal of template leads to recognition sites in molecularly imprinted polymer (MIP) that can selectively rebind the target.
  • MIP molecularly imprinted polymer
  • a copolymer of pyrrole and Bovine Serum Albumin is prepared as MIP. Electrical copolymerization of pyrrole and BSA is performed in perchlorate salt. Pyrrole monomers and albumin analytes are polymerized by giving alternating voltage using cyclic voltammetry.
  • MIP solution is prepared with 0.1M pyrrole and 0.5M lithium perchlorate (LiClO 4 ).
  • concentrations of MIP were prepared and listed in Table 1.
  • Table 1 0.1M pyrrole 0.5 M LiClO 4 BSA MIP10 2 mL 2 mL 2 mL 10 mg/mL MIP20 2 mL 2 mL 2 mL 20 mg/mL MIP30 2 mL 2 mL 2 mL 30 mg/mL MIP40 2 mL 2 mL 2 mL 40 mg/mL MIP50 2 mL 2 mL 2 mL 50 mg/mL MIP60 2 mL 2 mL 2 mL 60 mg/mL NIP 2 mL 2 mL None
  • the MIP solutions were agitated gently for 5 minutes and kept refrigerated for 1 hour.
  • a typical SPE 10 is shown in .
  • the MIP solution 12 is positioned and deposited using 1mm diameter tip on the working electrode 14 of the SPE.
  • the reference electrode 16 and counter electrode 18 used has copper wire submerged in the MIP solution.
  • the working electrode 14 is modified with the MIP.
  • the MIP has an area of 40 to 60 ⁇ m 2 .
  • the area can be set at 50 ⁇ m 2 .
  • the MIP sensor has three electrode that includes Ag/AgCl working and reference electrodes and MIP deposited carbon as working electrode.
  • the electrode is rinsed 5 times using deionized water to remove any remaining monomer and BSA analyte.
  • the cleaned electrodes are dried and stored in room temperature.
  • the device is adapted to be in contact with saliva and inserted in an Ampere reader to determine the level of albumin.
  • the slope or sensitivity of the curve is 0.33 mA mg -1 mL.
  • the limit of detection (LoD) of the MIP50 sensor is determined at 0.18 mg mL -1 .
  • the detection range of MIP50 is between 0.56 to 60 mg mL -1 .
  • MIP50 concentration has lowest LoD value at 0.18 mg mL -1 and also lowest LoQ at 0.56 mg mL -1 .
  • MIP20 concentration has the second lowest LoD and LoQ at 0.27 mg mL -1 and 0.82 mg mL -1 , respectively.
  • MIP60 has the highest LoD and LoQ values at 0.89 mg mL -1 and 2.89 mg mL -1 . It appears that MIP50 concentration has the best performance.
  • the MIP50 concentration sensor was tested against creatinine and urea.
  • the MIP50 concentration sensor has low reaction towards creatinine and urea.
  • the MIP50 concentration sensor has high selectivity, sensitivity and specificity for BSA.
  • the invention disclosed a device for determining albumin from saliva.
  • the device comprises of a modified SPE with a copolymer based on pyrrole and BSA.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
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  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The present invention provides a modified screen-printed electrode (SPE) for detecting albumin from saliva. The screen-printed electrode [10] has a counter electrode [18], a working electrode [14] and a reference electrode [16]. The working electrode [14] is modified with a molecularly imprinted polymer (MIP) [12] based on a copolymer of albumin analyte and pyrrole. The MIP [12] is derived from pyrrole, perchlorate salt and Bovine Serum Albumin. The SPE [10] is inserted in an Ampere reader to determine the level of albumin. Device sensitivity up to 0.33 mA mg-1mL is achieved with the device.

Description

DEVICE FOR DETECTING ALBUMIN FROM SALIVA
The present invention relates to a medical device for detecting protein, particularly a medical device based on electrochemical methods for detecting albumin from human saliva.
Albumin is a type of protein found in blood. Liver makes albumin. An albumin blood test is used to check the functions of liver and kidney. Albumin has a role in repairing tissue and helping the body grow while transporting vital hormones and nutrients around.
Abnormal albumin level could indicate a number of conditions such as liver disease, cirrhosis, kidney disease, malnutrition, infection, inflammatory bowel disease and thyroid disease.
A typical albumin blood test involves a thin needle to take blood from the vein. The blood is then filled in a tube. The tube is then sent to a lab for analysis. A protein electrophoresis machine is used to indicate the albumin level. It is a common procedure when a person undergoes a medical body check-up.
Henskens investigated salivary protein, albumin and cystatin concentrations. Shaila shown that salivary albumin was assessed using the Bromoscresol green method. The reaction between albumin in saliva and dye Bromoscresol green produces change in colour which is proportional to albumin concentration in saliva. The colour is estimated using a photoelectric colorimeter at wavelength of 630 nm.
US2010/0167306 disclosed a rapid test for glycated albumin in saliva. Saliva albumin is derived from plasma albumin and therefore contains glycated and non-glycated albumin fractions. The ratio of glycated albumin to total albumin in saliva is measured. The test kit is placed in a spectrophotometer or fluorometer to determine the ratio.
The albumin blood test is an invasive procedure that involves the drawing of blood. The procedure is time consuming as it needs to be sent to a lab with protein electrophoresis machine. Other described method also describes the use of machine to detect albumin. A simpler rapid test for detecting albumin without the use of machine is desired.
The present invention discloses a device for detecting albumin from saliva. A screen-printed electrode is modified to detect albumin. The screen-printed electrode has a counter electrode, a working electrode and a reference electrode. The working electrode is modified with a molecularly imprinted polymer (MIP) based on albumin analyte and pyrrole.
The MIP is derived from pyrrole, perchlorate salt and Bovine Serum Albumin. A drop of MIP was made on the working electrode. The area of MIP is 40 to 60 µm2.
The screen-printed electrode is made in contact with saliva. Then, it is inserted in an Ampere reader to determine the level of albumin
 Fig. 1
is a diagram of a modified surface-printed electrode according to the invention.
Fig. 2
is a diagram of current against the concentration of albumin.
 Detailed Description of Embodiments
Hereinafter, the present invention is described in detail.
Electrochemical sensors are gaining traction in instrumental analysis. Screen printing is a method to produce electrochemical sensors. This technology is used to prepare single use screen-printed electrodes (SPEs). SPEs surface can be functionalised to enhance electrochemical reactivity and sensitivity for specific analytes.
SPEs are attractive because it uses a small volume of samples. SPEs are also convenient as it is a form of in-situ test.
Molecular imprinting uses target as a template during polymerization of functional monomers. Subsequent removal of template leads to recognition sites in molecularly imprinted polymer (MIP) that can selectively rebind the target.
A copolymer of pyrrole and Bovine Serum Albumin (BSA) is prepared as MIP. Electrical copolymerization of pyrrole and BSA is performed in perchlorate salt. Pyrrole monomers and albumin analytes are polymerized by giving alternating voltage using cyclic voltammetry.
An example of MIP solution is prepared with 0.1M pyrrole and 0.5M lithium perchlorate (LiClO4). Various concentrations of MIP were prepared and listed in Table 1.
[Table 1] Table 1
0.1M pyrrole 0.5 M LiClO4 BSA
MIP10 2 mL 2 mL 2 mL 10 mg/mL
MIP20 2 mL 2 mL 2 mL 20 mg/mL
MIP30 2 mL 2 mL 2 mL 30 mg/mL
MIP40 2 mL 2 mL 2 mL 40 mg/mL
MIP50 2 mL 2 mL 2 mL 50 mg/mL
MIP60 2 mL 2 mL 2 mL 60 mg/mL
NIP 2 mL 2 mL None
The MIP solutions were agitated gently for 5 minutes and kept refrigerated for 1 hour.
Now, the surface imprinting process is described.
A typical SPE 10 is shown in . The MIP solution 12 is positioned and deposited using 1mm diameter tip on the working electrode 14 of the SPE. The reference electrode 16 and counter electrode 18 used has copper wire submerged in the MIP solution. The working electrode 14. The working electrode 14 is modified with the MIP.
The MIP has an area of 40 to 60 µm2. The area can be set at 50 µm2.
The potentiodynamic conditions followed during electropolymerisation are:
Potential range: -5.00V to 5.00V vs Cu
Scan rate: 50 mVs-1 with 5 current potential cycles
Using lithium perchlorate as solvent, hysteresis loop from cyclic voltammetry is achieved. This data is used to justify the formation of polypyrrole conductive polymer on SPE. The MIP sensor has three electrode that includes Ag/AgCl working and reference electrodes and MIP deposited carbon as working electrode.
After electropolymerization, the electrode is rinsed 5 times using deionized water to remove any remaining monomer and BSA analyte. The cleaned electrodes are dried and stored in room temperature.
The device is adapted to be in contact with saliva and inserted in an Ampere reader to determine the level of albumin.
shows linear relationship for MIP50 between current and BSA concentrations. The slope or sensitivity of the curve is 0.33 mA mg-1 mL. The limit of detection (LoD) of the MIP50 sensor is determined at 0.18 mg mL-1. The detection range of MIP50 is between 0.56 to 60 mg mL-1.
The performance and limit of quantitation (LoQ) of other MIP concentration is shown in Table 2.
[Table 2] Table 2
LoD
(mg mL-1)		 LoQ
(mg mL-1)		 Detection range
(mg mL-1)
NIP 0.31 0.94 0.94-60
MIP10 0.44 1.34 1.34-60
MIP20 0.27 0.82 0.82-60
MIP30 0.76 2.29 2.29-60
MIP40 0.36 1.08 1.08-60
MIP50 0.18 0.56 0.56-60
MIP60 0.89 2.89 2.89-60
It was observed that MIP50 concentration has lowest LoD value at 0.18 mg mL-1 and also lowest LoQ at 0.56 mg mL-1. MIP20 concentration has the second lowest LoD and LoQ at 0.27 mg mL-1 and 0.82 mg mL-1, respectively. MIP60 has the highest LoD and LoQ values at 0.89 mg mL-1 and 2.89 mg mL-1. It appears that MIP50 concentration has the best performance.
The MIP50 concentration sensor was tested against creatinine and urea. The MIP50 concentration sensor has low reaction towards creatinine and urea. Hence, the MIP50 concentration sensor has high selectivity, sensitivity and specificity for BSA.
Accordingly, the invention disclosed a device for determining albumin from saliva. The device comprises of a modified SPE with a copolymer based on pyrrole and BSA.
Non-patent citations
Henskens, Y. M. C., et al. "Protein, albumin and cystatin concentrations in saliva of healthy subjects and of patients with gingivitis periodonitis." Journal of periodontal research 28.1 (1993): 43-48.
Shaila, Mulki, G. Prakash Pai, and Pushparaj Shetty. "Salivary protein concentration, flow rate, buffer capacity and pH estimation: A comparative study among young and elderly subjects, both normal and with gingivitis and periodontitis." Journal of Indian society of periodontology 17.1 (2013): 42.

Claims (10)

  1. A device for detecting albumin from saliva, comprising:
    a screen-printed electrode [10] having a counter electrode [18], a working electrode [14] and a reference electrode [16];
    characterised in that,
    the working electrode [14] is modified with a molecularly imprinted polymer [12] based on albumin analyte and pyrrole.
  2. The device according to claim 1, wherein the molecularly imprinted polymer [12 is a derivation of pyrrole, perchlorate salt and Bovine Serum Albumin.
  3. The device according to claim 1, wherein the molecularly imprinted polymer [12 has an area of 40 to 60 µm2.
  4. The device according to claim 1, wherein the screen-printed electrode [10 is adapted to be inserted in an Ampere reader to determine the level of albumin.
  5. A method of preparing a device for detecting albumin, comprising:
    preparing a molecularly imprinted polymer solution [12] based on a copolymer of albumin analyte and pyrrole;
    preparing a screen-printed electrode [10] having a counter electrode [18], working electrode [14] and reference electrode [16]; and
    modifying the working electrode [14] with a drop of the molecularly imprinted polymer solution [12].
  6. The method according to claim 5, wherein the preparing of molecularly imprinted polymer [12 is made by copolymerizing pyrrole, perchlorate salt and Bovine Serum Albumin.
  7. The method according to claim 5, wherein the preparing of molecularly imprinted polymer [12 is made by copolymerizing pyrrole and Bovine Serum Albumin with alternating voltage using cyclic voltammetry.
  8. The method according to claim 5, wherein the modified working electrode [14 has a drop of molecularly imprinted polymer [12 with an area of 40 to 60 µm2.
  9. The method according to claim 5, further comprising submerging the reference electrode [16 and counter electrode [18 in the molecularly imprinted polymer solution [12.
  10. The method according to claim 5, further comprising inserting the screen-printed electrode [10 in an Ampere reader to determine the level of albumin.
PCT/MY2023/050086 2022-09-23 2023-10-24 Device for detecting albumin from saliva WO2024063638A1 (en)

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MYPI2022005228 2022-09-23

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060191788A1 (en) * 2001-11-26 2006-08-31 Ischemia Technologies, Inc. Electrochemical detection of ischemia
US20170122899A1 (en) * 2014-06-23 2017-05-04 The University Of Tokyo Sampling unit and biosensor
US20170241996A1 (en) * 2014-09-08 2017-08-24 Indian Institute Of Science Electrochemical biosensor and a method of sensing albumin and its complexes
US20190360959A1 (en) * 2017-02-09 2019-11-28 Agency For Science, Technology And Research A sensor
WO2020099560A1 (en) * 2018-11-16 2020-05-22 Danmarks Tekniske Universitet Electrochemical sensor system comprising molecularly imprinted polymer for early warning of urinary tract infections

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060191788A1 (en) * 2001-11-26 2006-08-31 Ischemia Technologies, Inc. Electrochemical detection of ischemia
US20170122899A1 (en) * 2014-06-23 2017-05-04 The University Of Tokyo Sampling unit and biosensor
US20170241996A1 (en) * 2014-09-08 2017-08-24 Indian Institute Of Science Electrochemical biosensor and a method of sensing albumin and its complexes
US20190360959A1 (en) * 2017-02-09 2019-11-28 Agency For Science, Technology And Research A sensor
WO2020099560A1 (en) * 2018-11-16 2020-05-22 Danmarks Tekniske Universitet Electrochemical sensor system comprising molecularly imprinted polymer for early warning of urinary tract infections

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