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WO2022034607A1 - Diagnostic de la covid-19 faisant intervenir une technologie laboratoire sur puce - Google Patents

Diagnostic de la covid-19 faisant intervenir une technologie laboratoire sur puce Download PDF

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Publication number
WO2022034607A1
WO2022034607A1 PCT/IN2021/050747 IN2021050747W WO2022034607A1 WO 2022034607 A1 WO2022034607 A1 WO 2022034607A1 IN 2021050747 W IN2021050747 W IN 2021050747W WO 2022034607 A1 WO2022034607 A1 WO 2022034607A1
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WIPO (PCT)
Prior art keywords
zno
electrode
covid
quantification
ace2
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Ceased
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PCT/IN2021/050747
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English (en)
Inventor
Arockiadoss THEVASAHAYAM
Krishnan MUTHUKALINGAN
Balasubramaniem ASHOK KUMAR
Varalakshmi Perumal
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • the present invention relates to medical diagnostic tool for detecting viral diseases. More particularly the present invention relates to a diagnosis and quantification tool for COVID- 19 employing LAB -ON-CHIP technology and method thereof.
  • CoV is an enveloped, positive-sense single-stranded RNA (ssRNA) virus belonging to the Coronaviridae family.
  • the CoV family consists of several species and causes upper respiratory tract and gastrointestinal infections in mammals and birds. In humans, it mainly causes common cold, but complications including pneumonia and SARS can occur.
  • the known human CoV includes HCoV-229E, -OC43, -NL63, - HKU1, and the more widely known severe acute respiratory syndrome coronavirus (SARS- CoV) which caused a global threat with high mortality in 2003.
  • the World Health Organization designated a sixth type of HCoV infection identified as the Middle East respiratory syndrome coronavirus (MERS-CoV) which is associated with high fatality.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • the main object of the present invention is to develop a rapid diagnosis and quantification tool for viral diseases in particular COVID- 19 employing LAB-ON-CHIP technology.
  • Figure 1 depicts block diagram of LIDS SENSO kit image with substrate and electrodes
  • Figure 2 depicts flowchart of mechanism and methods for diagnosis of COVID -19
  • 3a depicts the SEM images of electrode (Zn-ZnO- ACE2), With COVID 19 sample coated on electrode,
  • 3b depicts the SEM images of electrode (Zn-ZnO- ACE2), without COVID 19 sample coated on electrode,
  • 3c depicts the SEM image of Zn-ZnO nano rod on ACE2
  • Figure 4 depicts the Sensor results run by the LIDS SENSO diagnostic kit with COVID 19 samples.
  • Figure 5 depicts the LIDS SENSO DIAGNOSTIC KIT instrument and diagnostic kit image of the present invention.
  • Figure 6 depicts the Mobile apps final results.
  • the present invention discloses a rapid diagnosis and quantification tool for COVID- 19 employing LAB -ON-CHIP technology and method thereof.
  • the tool of the present invention comprises of base with two wells adapted to house two Zn-ZnO- ACE2 electrode. Each electrode comprises of two probes and one probe from the two electrodes are coupled with AC input and another probe from the two electrodes are coupled with circuit board/LIDS SENSO instruments capacitor output.
  • the tool further comprises of sample input for introducing the sample to the device.
  • This combination of SARS CoV-2 with ACE2 receptor results in change in capacitance (as a Key point) which can be measured with another 2 probes for the identification as well as quantification of CO VID 19.
  • the present invention discloses a rapid diagnosis and quantification tool for COVID- 19 employing LAB -ON-CHIP technology and method thereof.
  • Micro-fluidics on physics manipulation and study of minute amounts in lab-on-a-chip technology is depends on at least two major scientific disciplines - micro fluidics, and molecular biology. Nanotechnology play a key role in tying these two fields together as the technology progresses the application of the principle of electronics to Biology and medicine.
  • the Process of specific analyte (Receptor/ Antibody) binding to the probe molecule (Antigen) immobilized (Agglutination) on the electrode for detection of Receptor/Antigen and Antibody interactions is specific chemical interaction between antibodies produce by B cells of the white blood cells and antigens during immune reaction.
  • the antigens and antibodies combines by a process called agglutination.
  • Antigens are bond to antibodies through weak and noncovalent bonds, Vander Waals forces, and hydrophobic interactions.
  • Human coronovirus NL63 is a species of coronovirus that was identified in late 2004.
  • the virus is an enveloped positive - sense, single stranded RNA virus (Severe Acute Respiratory Syndrome (SARS-CoV-2) spike protein S which enter in host cells by the angiotensin - Converting Enzyme -2 (ACE-2).
  • SARS-CoV-2 spike protein (s) has N- terminus responsible for receptor binding of N/C terminal region (aal to 400) of different ACE-2 protein.
  • ACE 2 is a zinc containing metalloenzyme located on the surface of endothelial and other cells. It contains N-terminal peptides M2 domain and a C -terminal collection renal amino acid transporter domain. ACE 2 is a single-pass type 1 membrane protein, with its enzymatically active domain exposed on the surface of cells in lungs and other tissues. The extra cellular domain of ACE2 is cleaved from the transmembrane domain by another enzyme known as SHEDDASE and the resulting soluble protein is released into the blood and ultimately excreted into urine.
  • SHEDDASE another enzyme
  • Zn-ZnO nano rod preparation In an aspect of the present invention, crystalline ZnO nanorods are prepared by hydrothermal method with controlled condition of pressure, temperature and catalysts. The size, length and purity level along with morphology of the ZnO nanorod growth are optimized. The nanorods are grown on Zn plate using ammonia (NH3OH) and Hydrogen peroxide (H2O2) equal ration (1:1) according to the level of the substrate for 70 to 100 °C for 12 hrs. The ZnO nano rod on Zn plate was removed and washed with distilled water three to four times. The Zn-ZnO nanorods are subsequently used for fictionalization.
  • NH3OH ammonia
  • H2O2O2 Hydrogen peroxide
  • the Zn -ZnO nanostructures are immersed in the solution of N-(15- carboxy pentadecano yloxy) succiniimide (NHS) in of ethanol and butanol in the ratio of 2:1 along with 2-(N-morpholine)-ethane sulfonic acid (MES) in deionized water and l-ethyl-3- (3 -dimethylaminopropyl) (EDC) (Linker) to form a precursor (Zn-ZnO- EDC) for attachment of Angiotensin Converting Enzyme II (ACE-2).
  • NHS N-(15- carboxy pentadecano yloxy) succiniimide
  • MES 2-(N-morpholine)-ethane sulfonic acid
  • EDC l-ethyl-3- (3 -dimethylaminopropyl)
  • Linker to form a precursor (Zn-ZnO- EDC) for attachment of Angiotensin
  • Zn-ZnO-ACE-2 elelctrode preparation The above nano structure of Zn-ZnO- EDC After 30 minutes 2 to 10 nano liter (according to the covid 19 virus concentration) of Angiotensin Converting Enzyme II (ACE-2) is mixed with 20 ml Phosphate Buffer for 10 to 15 minutes. The mixture is added (Zn-ZnO-EDC) and leave for 30 minutes and then washed with deionized water and stored in 4 °C finally Zn-ZnO- ACE2 electrode is ready for diagnosis and quantification of COVID-19/SARS CoV-2.
  • ACE-2 Angiotensin Converting Enzyme II
  • LIDS SENSO kit preparation The above Electrode Zn-Zno- ACE2 is inserted in the prepared kit using poly acrylic sheet Figurel. Then, the biomolecule (Np/No-swab or blood) sample collected from patient in the 2 to 3 ml isotonic solution or phosphate buffer solution from this 500 micro liter is taken and added in the LIDS SENSO kit. Then keep is in the automatic sensor setup.
  • the present invention discloses a rapid diagnosis and quantification tool for COVID-19 employing LAB-ON-CHIP technology and method thereof.
  • the tool of the present invention comprises of base with two wells adapted to house two Zn-ZnO- ACE2 electrode. Each electrode comprises of two probes and one probe from the two electrodes are coupled with AC input and another probe from the two electrodes are coupled with circuit board/LIDS SENSO instruments capacitor output.
  • the tool further comprises of sample input for introducing the sample to the device.
  • This combination of SARS CoV-2 with ACE2 receptor results in change in capacitance (as a Key point) which can be measured with another 2 probes for the identification as well as quantification of CO VID 19.
  • the present invention shall disclose a method for rapid diagnosis and quantification for COVID-19 employ the developed tool of the present invention.
  • the method comprising of introducing a sample to be tested in the tool and measuring the change in capacitance as a key point to detect as well as quantify the CO VID 19.
  • the results can be viewed either using Bluetooth based data to mobile apps or system based internal storage PC/Labtop.
  • the sample comprising of blood or throat swap or nasal swab and the like.
  • the time for detection and quantification of COVID 19 employing the tool of the present invention is 18 (6 +12) seconds, may vary the time period according to the concentration of virus.
  • the developed rapid diagnosis and quantification tool for COVID-19 was next subjected to clinical trials to find the efficacy of the device.
  • FIG 3a depicts the SEM images of electrode (Zn-ZnO- ACE2), With COVID 19 sample coated on electrode.
  • the first area is uncoated with COVID-19 sample and second area is coated for identification and confirmation by scanning electron microscopic (SEM) long view (1000 micron).
  • SEM scanning electron microscopic
  • 3b depicts the SEM images of electrode (Zn-ZnO- ACE2), without COVID 19 sample coated on electrode, The first area is uncoated with COVID-19 sample and second area is coated for identification and confirmation by scanning electron microscopic (SEM) closure view (2 micron).
  • SEM scanning electron microscopic
  • 3c depicts the SEM image of ZnO nano rod bonded with ACE2 as per chemical procedure to ensure the presents of EDC on Zn-ZnO nano rod and 3d depicts the ED AX spectrum of Zn- ZnO- ACE2 - Cov-2 to ensure the chemicals as per the virus sample here the virus sample present in the Viral Transport Medium (VTM), hence the phosphate and chlorine peaks are present.
  • VTM Viral Transport Medium
  • FIG. 4 depicts the Sensor results run by the LIDS SENSO diagnostic kit with COVID 19 samples.
  • the results are represent, 1, Initially (2 to 101 scale of x-axis) we run without any sample with senso kit, 2. Only Viral Transport Medium (VTM) for ensuring the interaction with senso kit (Zn-ZnO-EDC) and medium (102 to 201 scale of x-axis), after that the VTM sample with patient samples (202 to 701 scale of x-axis) - the peaks from 202 301are COVID -19 positive and the peaks from 302 to 401 are negative COVID -19 sample, the peaks from 402 to 601 the second and third positive COVID samples respectively, and the peaks from 602 to 702 are the second negative sample.
  • VTM Viral Transport Medium
  • Figure 5 depicts the LIDS SENSO DIAGNOSTIC KIT instrument and diagnostic kit image of the present invention and Figure 6 depicts the Mobile apps final results thereby proving user friendly of the kit of the present invention
  • the present invention shall disclose a rapid diagnosis and quantification tool for detecting viral strain employing LAB -ON-CHIP technology.
  • the rapid diagnosis and quantification tool comprises of a base with two wells adapted to house two Zn-ZnO- ACE2 electrode and sample input provision adapted to receive the sample and transfer to the electrode in which each electrode comprises of two probes, one probe from the two electrodes are coupled with AC input adapted to transmit an alternating current of peak to peak 4 Volts with AC frequency of 100Hz to 1 KHz in particular 500 Hz and another probe from the two electrodes are coupled with circuit board/LIDS SENSO instruments capacitor output adapted to measure change in capacitance upon combination of Viral Strain with ACE2 receptor by attraction of N-terminal spike protein of Viral Strain by N- terminal of ACE2 receptor for identification as well as quantification of viral diseases.
  • the Viral strain is selected from a group comprising of SARS CoV-2, as per the ICMR India prescribed solution Viral Transport Medium (VTM)/ /Isotonic solution.
  • VTM Viral Transport Medium
  • the present invention shall disclose a rapid diagnosis and quantification tool for COVID-19 employing LAB-ON-CHIP technology.
  • the rapid diagnosis and quantification tool comprises of a base with two wells adapted to house two Zn-ZnO- ACE2 electrode and sample input provision adapted to receive the sample and transfer to the electrode.
  • Each electrode comprises of two probes, one probe from the two electrodes are coupled with AC input which is adapted to transmit an alternating current of peak to peak 4 Volts with AC frequency of 100Hz to 1 KHz in particular 500 Hz and another probe from the two electrodes are coupled with circuit board/LIDS SENSO instruments capacitor output and is adapted to measure change in capacitance upon combination of SARS CoV-2 with ACE2 receptor by attraction of N-terminal spike protein of SARS CoV-2 by N- terminal of ACE2 receptor for identification as well as quantification of COVID 19.
  • the Zn-ZnO- ACE2 electrode is prepared by a process comprising of following steps: a) Preparing Zn-ZnO nano rod comprising of growing nanorods on Zn plate using ammonia and Hydrogen peroxide in equal ratio according to level of substrate for 70 to 100 °C for 12 hrs followed by removing Zn-ZnO nano rod from Zn plate and washing with distilled water; b) fictionalization of the Zn-ZnO nano rod to form Zn-ZnO- EDC by immersing the said Zn-ZnO nano rod in a solution comprising of ethanolic N-(15- carboxy pentadecano yloxy) succiniimide (NHS) and butanol in a ratio of 2:1 along with 2-(N-morpholine)-ethane sulfonic acid (MES) in deionized water and l-ethyl-3-(3-dimethylaminopropyl) (
  • the circuit board/LIDS SENSO instruments capacitor output comprises of arudino board, dot broad ( de to ac frequency converter) and blue tooth sensor board with adequate home design software for control stepper motor and adapted to send and receive capacitance signals.
  • the present invention shall disclose a method for rapid diagnosis and quantification for COVID-19 employing rapid diagnosis and quantification tool of the present invention.
  • the method comprises of introducing a sample to be tested in sample input provision and measuring the change in capacitance to detect as well as quantify the COVID 19 in which the capacitance can be viewed either using Bluetooth based data to mobile apps or system based internal storage PC/Labtop.
  • the sample is selected from blood or throat swap or nasal swab and the like.
  • the diagnosis and quantification for COVID-19 is carried out within a time period of 18 to 60 seconds.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Physics & Mathematics (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)

Abstract

La présente invention concerne un outil de diagnostic rapide et de quantification pour la COVID-19 faisant intervenir la technologie laboratoire sur puce et son procédé. L'outil de la présente invention comprend une base comprenant deux puits adaptés pour loger deux électrodes Zn-ZnO-ACE2. Chaque électrode comprend deux sondes et une sonde à partir des deux électrodes sont couplées à une entrée AC et une autre sonde depuis les deux électrodes sont couplées à une carte de circuit/sortie de condensateur d'instruments LIDS SENSO. L'outil comprend en outre une entrée d'échantillon pour introduire l'échantillon au niveau du dispositif.
PCT/IN2021/050747 2020-08-08 2021-08-04 Diagnostic de la covid-19 faisant intervenir une technologie laboratoire sur puce Ceased WO2022034607A1 (fr)

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IN202041034067 2020-08-08

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018208610A1 (fr) * 2017-05-11 2018-11-15 Arizona Board Of Regents On Behalf Of Arizona State University Appareil de point d'intervention et procédés de détection d'analyte à l'aide d'une impédance ou d'une capacité électrochimique
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Patent Citations (3)

* Cited by examiner, † Cited by third party
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WO2018208610A1 (fr) * 2017-05-11 2018-11-15 Arizona Board Of Regents On Behalf Of Arizona State University Appareil de point d'intervention et procédés de détection d'analyte à l'aide d'une impédance ou d'une capacité électrochimique
CN111474350A (zh) * 2020-04-23 2020-07-31 中国林业科学研究院林业研究所 一种检测冠状病毒s1抗原的试剂盒及其非诊断目的的检测方法
CN111443072A (zh) * 2020-04-30 2020-07-24 苏州翊清环境科技有限公司 病毒检测用拉曼芯片、制备方法、及病毒快速检测方法

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