FIELD OF THE INVENTION:

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.

BACKGROUND OF THE INVENTION:

At the end of December 2019, Chinese public health authorities reported several cases of acute respiratory syndrome in Wuhan City, Hubei province, China. Chinese scientists soon identified a novel coronavirus as the main causative agent. The disease is now referred to as coronavirus disease 2019 (COVID-19), and the causative virus is called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is a new strain of coronavirus that has not been previously identified in humans. The initial outbreak in Wuhan spread rapidly, affecting other parts of China. Cases were soon detected in several other countries. Outbreaks and clusters of the disease have since been observed in Asia, Europe, Australia, Africa and the Americas.

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. [Van der Hoek etal.,2007] The known human CoV (HCoV) 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. In 2012, the World Health Organization (WHO) designated a sixth type of HCoV infection identified as the Middle East respiratory syndrome coronavirus (MERS-CoV) which is associated with high fatality. As of 16 July 2020, 13,776,752 cases of COVID-19 were reported worldwide by more than 213 countries and territories. Since late February, the majority of cases reported are from outside China, with an increasing majority of these reported from EU/EEA countries and the UK. The Director General of the World Health Organization declared COVID-19 a global pandemic on 11 March 2020.

The COVID-19 outbreak has had a major impact on clinical microbiology laboratories in the past several months. This commentary covers current issues and challenges for the laboratory diagnosis of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2). In the preanalytical stage, collecting the proper respiratory tract specimen at the right time from the right anatomic site is essential for a prompt and accurate molecular diagnosis of COVID- 19. Appropriate measures are required to keep laboratory staff safe while producing reliable test results. In the analytic stage, real-time reverse transcription-PCR (RT- PCR) assays remain the molecular test of choice for the etiologic diagnosis of SARS-CoV-2 infection while antibody-based techniques are being introduced as supplemental tools. In the post analytical stage, testing results should be carefully interpreted using both molecular and serological findings. Finally, random-access, integrated devices available at the point of care with scalable capacities will facilitate diagnosis and monitoring of SARS-CoV-2 infections. (Yi-Wei Tang et al 2020).

All the available diagnostic kits and method for detecting COVID 19, either often shows false positive and negative results and none of the available techniques demonstrates quantification of the diseases. Thus there exists an urgent need in the state of art to develop a novel tool and technique which both, diagnosis and quantify COVID- 19.

OBJECT OF THE PRESENT INVENTION:

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.

Another object of the present invention is to develop a LAB -ON-CHIP technology based on attraction of SARS CoV-2 with ACE2 receptor applying Alternating Current of peak to peak 4 volts with 100 to 1 KHz and measuring the change in capacitance, before and after the binding of SARS COV-2 RBD to ACE-2 receptor (to activate by an applied AC frequency) for both diagnosis and quantification of COVID - 19. Further object of the present invention is to employ the developed technology for diagnosis and quantification of COVID- 19 in the level of infection and to identification/diagnostic of other virus present in the biomolecules.

BRIEF DESCRIPTION OF DRAWINGS:

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

Figure 3 depicts

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 and

3d depicts the ED AX spectrum of Zn-ZnO- ACE2 - Cov-2

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.

SUMMARY OF THE INVENTION:

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. The two probes coupled with AC input on applying an alternating current of peak to peak 4 Volts with frequency of 100Hz to 1 KHz in particular 500 Hz according to the concentration of ACE2 receptor, attracts the N-terminal spike protein of SARS CoV-2 by N- terminal of ACE2 receptor. 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.

DETAILED DESCRIPTION OF THE INVENTION:

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.

The potential N -Glycosylation sites are indicated by the crucial amino acid residues that are involved in direct contact between huACE-2 and SARS-CoV-2 S protein of asparagines residues. 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.

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.

For fictionalization, 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).

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.

LIDS SENSO diagnostic Instrument preparation:

The instrument compressing arudino board and dot broad ( de to ac frequency converter) and blue tooth sensor board with adequate home design software for control the stepper motor and send and receive the signals (capacitance) from the diagnostic kit. 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.

Diagnostics and quantifications of COVID -19: The senso setup switched on after keeping the above LIDS senso kit, initially 6 seconds for reaching the four probe in the electrode and immediately apply the peak to peak 4 Volts with AC frequency of 100Hz to 1 KHz in particular 500 Hz for 12 minutes to attaching / binding the COVID- 19/SARS CoV to the ACE2 receptor and simultaneously we can measure the capacitance of the electrode/LIDS SENSO kit. After that the probes are removed and the LIDS SENSO kit dispose proper way. The binding of biomolecules to the complex is confirmed by the conventional methods such as microscopy, Polymerase Chain Reaction (PCR), absorbance and scattering method.

In one aspect, 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. The two probes coupled with AC input on applying an alternating current of peak to peak 4 Volts with AC frequency of 100Hz to 1 KHz in particular 500 Hz according to the concentration of ACE2 receptor, attracts the N-terminal spike protein of SARS CoV-2 by N- terminal of ACE2 receptor. 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.

In another aspect 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. As per the invention, the sample comprising of blood or throat swap or nasal swab and the like. In accordance with the invention, 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.

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).

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).

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. This is another confirmation of technology.

Figure 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. The above results are confirmed by the RT-PCR test simultaneously.

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 In most preferred embodiment, 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.

As per the invention, 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.

In one of the preferred embodiment, 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.

According to the invention, in the rapid diagnosis and quantification tool, 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) (EDC) (Linker) to form a precursor (Zn-ZnO- EDC); c) preparing Zn-ZnO- ACE-2 electrode comprising of mixing 2 to 10 nano liter of Angiotensin Converting Enzyme II (ACE-2) with 20 ml Phosphate Buffer for 10 to 15 minutes followed by adding the (Zn-ZnO-EDC) and allowed to stand for 30 minutes and finally washed with deionized water and stored in 4 °C to form the Zn-ZnO- ACE2 electrode

In accordance with the invention, in the rapid diagnosis and quantification tool, 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.

In another preferred embodiment, 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.

In accordance with the invention, in the method, the sample is selected from blood or throat swap or nasal swab and the like.

According to the invention, in the method, the diagnosis and quantification for COVID-19 is carried out within a time period of 18 to 60 seconds.

While considerable emphasis has been placed herein on the various components of the preferred embodiment, it will be appreciated that many alterations can be made and that many modifications can be made in the preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiment as well as other embodiments of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.