WO2022038636A1

WO2022038636A1 - A diagnostic device and method for differentiating asthma-copd overlap syndrome (aco) from asthma and copd

Info

Publication number: WO2022038636A1
Application number: PCT/IN2021/050793
Authority: WO
Inventors: Gyanendra Singh; Souvik Biswas; Arijit PAL; Nilanjana GHOSH; Parthasarathi Bhattacharyya; Koel Chaudhury
Original assignee: Indian Council Of Medical Research; Indian Institute of Technology Kharagpur
Priority date: 2020-08-19
Filing date: 2021-08-17
Publication date: 2022-02-24
Also published as: KR20230049724A

Abstract

The present invention provides a device and method for differentiating Asthma-COPD Overlap (ACO) from Asthma and COPD. SAM layer formed on gold electrodes is characterized in the experimental set up. The mounting of antibody on activated gold electrode is confirmed through various assays. The fabrication of the device is standardized for reproducibility of the data with IL-4 Antibody.

Description

A diagnostic device and method for differentiating Asthma-COPD Overlap Syndrome (ACO) from Asthma and COPD

TECHNICAL FIELD

The present invention relates to an IL-4 based SAM modified impedimetric analytical device and a method for differentiating Asthma-COPD Overlap (ACO) from Asthma and Chronic obstructive pulmonary disease (COPD). The present invention provides an electrochemical immunosensor and a method for preparing electrochemical immunosensor on a gold electrode for early and accurate diagnosis of Asthma-COPD Overlap Syndrome (ACO).

BACKGROUND

Asthma is an obstructive lung disease, usually characterized by chronic airway inflammation. These patients have variable expiratory airflow limitation accompanied by a history of symptoms such as cough, wheezing, chest tightness and shortness of breath (1). Chronicobstructive pulmonary disease (COPD), is characterized by persistent airflow limitation which is progressive in severity. It is associated with chronic airway inflammation linked to noxious particles or gases like tobacco smoke (2). Asthma-COPD overlap (ACO) refers to patients presenting with characteristics of both asthma and COPD. Gibson and Simpson proposed the term asthma COPD overlap syndrome (ACO) in 2009 to describe patients with features of both, asthma and COPD (Gibson et al., 2009). ACOS was later replaced with ACO, by the global bodies of GINA and GOLD as the former was often misinterpreted as a single disease entity. COPD overlap syndrome (ACOS) as asthma-COPD overlap (ACO). Therefore, the inventors are using abbreviation (ACO) for referring to asthma-COPD overlap syndrome in the present specification. These patients experience a more rapid decline in lung function, have poor quality of life, experience frequent exacerbations, have increased mortality and consume more resources than asthma or COPD alone (3-5). There are many cases of pathologic and functional overlap between asthma and COPD, yet authorities debate whether this overlap represents the coexistence of two common airway diseases or whether there are common underlying pathogenic mechanisms leading to this common phenotype.The global bodies of GOLD and GINA jointly, have renamed asthma-

The controversy on how to distinguish asthma from COPD has lasted for decades, debating whether it is asthma or COPD alone, or if there is another possibility that links the above two diseases. The two well-known hypotheses are British hypothesis and Dutch hypothesis, but neither has been confirmed nor refused to date (6). Both asthma and COPD are inflammatory diseases with asthma generally associated with eosinophilic airway inflammation and COPD with neutrophilic inflammation. However, it has been observed that eosinophilia, neutrophilia and a mixed inflammatory phenotype can exist in both. There are a large number of different immunological and inflammatorymarkers which are altered in asthma and COPD, some of which are common and some unique making these diseases difficult to classify (7-8). The concept of the ACO has emerged with increasing recognition of the heterogeneity of both asthma and COPD. The airway inflammation pattern in ACO is associated with 35% eosinophilic and 19% neutrophilic bronchitis respectively, with 10% showing a mixed inflammatory pattern. These data further proof that ACO is a heterogeneous inflammatory disorder of the airways.

While some studies suggest that ACO maybe a distinct phenotype in patients with adult asthma others have reported COPD like neutrophil-dominant inflammation in ACO (9-12). Reports have shown that immunological mediators like interleukin-6 (IL-6), C-reactive protein (CRP), tumour necrosis factor-a (TNF- a ) and surfactant protein A (SP-A) are elevated in ACO patients (13- 14). Recently, expressions of 7 types of cytokines (IL-4, IL-5, IL-8, IL-9, IL- 13, IL-17A and IL- 27), and 6 angiogenesis related factors (vascular endothelial growth factor A (VEGFA), VEGFC, VEGFD, basic fibroblast growth factor (bFGF), Fms-like tyrosine kinase 1 (FLT-1) and placental growth factor (PIGF)) have been determined to evaluate the differential diagnostic potential of serum biomarkers in ACO. In another study, IL-4, IL-8, and IL- 10, and TNF-a levels have been measured in patients with asthma, acute exacerbation of COPD (AECOPD), and ACO and statistically significant differences were observed among the three groups.

Overlap syndrome appears to share many of the same disease risk factors as that of asthma and COPD. Since ACO patients are excluded from randomized controlled trials, there is an absence of evidence for treatment recommendations in this group. It appears from the above cited literature that suitable end point is missing in characterizing ACO from other respiratory diseases. It is evident that there is a need to identify a biomarker inexpensive, portable, and easy- to-operate, point of care diagnostic tool for early and effective diagnosis of this airway disease. It would be a stepping stone towards the development of an individually tailored therapeutic approach, popularly termed as 'personalized molecular biology’. Clinical need:

Asthma is a respiratory disease, heterogeneous in nature and commonly characterized by chronic airway inflammation and obstruction. It is characterized by a series of respiratory symptoms such as sneezing, shortness of breath, chest tightness followed by cough that gets intensified with progression of disease with time (1). It is known to be initiated by a combination of genetic and environmental factors and is not limited only to exposure to air pollution and allergen. The chronic airflow limitation characteristic of COPD is caused by a mixture of mucus hypersecretion (chronic bronchitis) and parenchymal destruction (emphysema). The term “Asthma- COPD Overlap" has been recently defined and is being debated as a new pathological entity. It is characterized by persistent airflow limitation with several features usually associated with asthma and several features usually associated with COPD. ACO is therefore identified by the features that it shares with both asthma and COPD (1-2). Local and systemic responses are highly activated in both asthma and COPD and is well elucidated through different studies. In recent years, ACO has generated considerable interest amongst researchers and various immunological markers in the disease have been investigated.

The significant heterogeneity existing in the airways has drawn more attention towards ACO. Compared with asthma or COPD patients, ACO shows an increased disease burden with greater number of hospitalizations, increased exacerbations and health status impairment. ACO patients are generally excluded from randomized controlled trials owing to the ambiguity associated with the disease phenotype. Consequently, treatment recommendations for ACO remain unclear. Hence, there is an absolute need for characterizing ACO at a molecular level to understand this overlap disease pathophysiology. Apart from this, there is an absolute need for the development of indigenous system and method for differentiating Asthma-COPD Overlap (ACO) from Asthma and COPD. This could assist in the development of appropriate therapies for better health care management.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified format that are further described in the detailed description of the invention. This summary is not intended to identify key or essential inventive concepts of the claimed subject matter, nor is it intended for determining the scope of the claimed subject matter.

In accordance with the present subject matter, it is an objective of the present disclosure to differentiate Asthma COPD overlap (ACO) from Asthma and Chronic Obstructive Pulmonary Disease (COPD). In one of the embodiment of the present invention, it provides a process for preparing electrochemical immunosensor on a gold electrode for early and accurate diagnosis of Asthma- COPD Overlap Syndrome (ACO).

In another embodiment of the present invention, it provides an electrochemical immunosensor on a gold electrode for early and accurate diagnosis of Asthma-COPD Overlap Syndrome (ACO) by detecting antigen antibody complex formation in ACO associated COPD and converting it to an electrical signal for processing, recording and display; wherein ACO marker is Interleukin-4 (IL- 4).

To further clarify advantages and features of the present invention, a more particular description of the invention is rendered by reference to specific embodiments thereof in the detailed description.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

To further clarify advantages and aspects of the invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in accordance with various embodiments of the invention, wherein:

Figure 1: Schematic representation of the different fabrication stages

Figure 2: FTIR Spectra of 3 Mercaptopropionic acid ethanolic solution

Figure 3: FTIR Spectra for verification of SAM of 3-MPA on the gold surface

Figure 4: ATR-FTIR spectra for different concentrations of 3 -mercaptopropionic acid (3-

MPA) in ethanol namely (a) lOmM (b) 20mM (c) 30mM and (d) 40mM respectively

Figure 5: ATR-FTIR spectra for SAM layer formation on gold electrodes at (a) lOmM (b) 20mM and (c) 30mM (d) 40mM concentration of 3-mercaptopropionic acid in ethanol

Figure 6: AFM images of SAM layer using (a) lOmM (b) 20mM (c) 30mM and (d) 40mM of 3-MPA in ethanol

Figure 7: Comparison between (a) thickness and (b) roughness of different SAM layers using AFM

Figure 8: Electrochemical verification of SAM formation using Impedance Spectroscopy

Figure 9: EIS plot of EDC NHS ester formation

Figure 10: EIS plot after SAM bound Antibody Figure 11: EIS plot for antigen-antibody interaction

Figure 12: EIS plot for comparing different stages of sensor fabrication after gold surface activation with EDC and NHS

Figure 13: Verification of antibody binding by coupling the HRP tagged antibody on the EDC/NHS activated SAM on gold electrode and observing the colorimetric change by adding TMB substrate.

For better understanding the figures have been modified and rearranged. A separate word file is provided for the figures with better image resolution.

It may be noted that to the extent possible, like reference numerals may have been used to represent like elements in the drawings. Further, those of ordinary skill in the art will appreciate that elements in the drawings are illustrated for simplicity and may not have been necessarily drawn to scale. For example, the dimensions of some of the elements in the drawings may be exaggerated relative to other elements to help to improve understanding of aspects of the invention. Furthermore, one or more elements may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details that are pertinent to understanding the embodiments of the invention so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

DETAILED DESCRIPTION:

For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are explanatory of the invention and are not intended to be restrictive thereof.

Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment. The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.

In one of the embodiment of the present invention, it provides a process for preparing electrochemical immunosensor on a gold electrode for early and accurate diagnosis of Asthma- COPD Overlap Syndrome (ACO), said method comprising:

(a) preparing a self- assembled monolayer layer (SAM) on gold electrode by: i. exposing gold electrodes to oxygen plasma; ii. rinsing the electrode with alcohol and double distilled water successively and drying in nitrogen to obtain pre treated electrode; iii. immersing the pretreated electrode in ethanol solutions of 3- mercaptopropanoic acid (3 -MPA) at four different concentrations lOmM, 20mM, 30mM and 40mM, respectively and keeping the electrodes in the solution for 24 hours, at room temperature and controlled humidity conditions, to form a self assembled monolayer (SAM); iv. after ~24hrs incubation at room temperature, the substrates are removed and rinsed by squirting excess ethanol over them to remove unbound thiols and the substrates are blown dry with nitrogen;

(b) activation of the self-assembled monolayer layer (SAM) on gold electrode; i. treating the thiol-modified electrodes with 0.4 mM.EDC-0.lmM NHS for 15mins to 1 hour to convert the terminal carboxylic groups to an active NHS ester; (c) antibody attachment on surface of the activated self-assembled monolayer layer (SAM) on the gold electrode; i. dropping 10-20 ng/ml of Anti-Human IL-4 onto the surface of the activated SAM at 37 °C PBS for 1-2 hr; ii. removing excess antibodies by rinsing with PBS; iii. treating the antibody-modified electrodes with 0.1-0.5% BSA for 30 mins to 1 hour, unreacted and non-specific sites; iv. after rinsing with PBS and water, drying the electrodes under nitrogen;

(d) performing impedance measurements after gold surface activation with EDC and antibody immobilization; and

(e) after Ab immobilization, non-specific binding was blocked using BSA.

In another embodiment of the present invention, it providesan electrochemical immunosensor on a gold electrode for early and accurate diagnosis of Asthma-COPD Overlap Syndrome (ACO) by detecting antigen antibody complex formation in ACO associated COPD and converting it to an electrical signal for processing, recording and display; wherein ACO marker is Interleukin-4 (IL- 4).

In yet another embodiment of the present invention, it provides the immunosensor, wherein said immunosensor differentiates ACOfrom Asthma and COPD.

In yet another embodiment of the present invention, it provides the immunosensor, wherein the immunosensor provides output within one minute at room temperature using 50 pl of serum sample of the subject.

In another embodiment of the present invention, it provides a method for differentiating Asthma-

COPD Overlap Syndrome (ACO) from Asthma and COPD, said method comprising: preparing and activating a SAM layer on gold electrodes; and mounting antibody on activated gold electrode; wherein the Interleukin-4 (IL-4) is an immune mediator and the immobilized antibody binds to the IL-4 present in the serum of the subject.

Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible. EXAMPLES

The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Design:

This is part of an ongoing collaborative study between IIT Kharagpur and Institute of Pulmonary and research (IPCR), Kolkata. Ethical clearance and informed consent had been obtained prior to the commencement of the study. Samples were obtained from patients reporting for pulmonary ailments at IPCR Kolkata and from healthy individuals also. Asthma, COPD and ACO patients were recruited as per prescribed global guidelines. Serum aliquots (-100 pl) stored in sterile cryovials at -80°C were used for experimentation purpose. An array of immune mediators has been studied to explore whether these markers can effectively distinguish ACO from 'pure' asthma and 'pure' COPD. Indirect ELISA is being employed to study these markers which include Interleukin 4 (IL-4), Interleukin 13 (IL-13), Interleukin 6 (IL-6), Interleukin 17 (IL-17), Tumor Necrosis Factor- a (TNF-a), Monocyte Chemoattractant Protein- 1 (MCP-1); Interleukin 8 (IL-8), Transforming Growth Factor-P (TGB-P), C-Reactive Protein (CRP), Eosinophilic Cationic Protein (ECP), Neutrophil Gelatinase- Associated Lipocalin (NGAL) and Surfactant Protein A (SP-A). Further, the serum levels of these 12 immunological mediators were measured using commercially available ELISA kits (Quantikine; R & D Systems Inc., Minneapolis, MN, USA). The test procedure as per the manufacturer’s instruction was adopted. IL-4 and MCP-1 showed the most promising results; IL-4 was selected for sensor development since it is reported to be highly dysregulated in ACO.

The affinity of the above array of serum biomarkers has been tested to help define and segregate ACO. The second phase work involves development of paper based ELISA assay for the antigen/ biomarker identified to be significantly altered in ACO followed by the fabrication of the device in terms of biosensors with the ongoing need of quick diagnosis with more sensitivity. Our study indicates that IL-4 is the most promising marker for differentiating ACO from asthma and COPD. This further motivated us to develop a diagnostic platform for differential diagnosis of ACO. The objective being, development of an IL-4 based SAM modified impedimetric analytical platform, that can be used to detect the antigen in serum. The analytical platform was developed keeping in mind technical feasibility even in conditions of limited medical infrastructure, simplicity of usage by the end user, limited sample requirement and short time involvement for obtaining results. It is envisaged that this technology could be useful to the clinicians as an alternate for ACO screening and differential diagnosis. The platform has been developed with an aim of providing quick diagnosis accompanied with high sensitivity and specificity.

Biosensors are compact analytical devices used for the detection of specific analytes and have gained considerable popularity in the field of medical diagnosis. Immunosensors capable of detecting antigen antibody complex formation in ACO associated COPD and subsequently converting it to an electrical signal for further processing, recording and display has been attempted. Amidst the variety of biosensors available, immunosensors have seen an increase in growth over the last decade. This may be attributed to the fact that there is a demand for sensitive, fast, portable, cost-effective and easy-to-use devices for the detection of biomarkers (15). Studies on immunosensors in lung cancer are documented. Aydin et al. (2017) developed a sensitive, disposable indium tin oxide (ITO)-based electrochemical immunosensor for simple, rapid determination of sex-determining region Y-box 2 (SOX2), a marker for lung adenocarcinoma (16). Another study discusses fabrication of a label-free electrochemical immunosensor for the detection of cytokeratin 19 fragment antigen 21-1 (CYFRA21-1), a marker for non- small cell lung cancer (17). Kim et al. (2009) have developed an amperometric immunosensor for detection of Annexin II and MUC5AC which are considered to be promising markers of lung cancer (18). However, no immunosensors have been reported for ACO to differentiate between Asthma and COPD. Hence, attempts have been made to develop protein based diagnostic sensor for minimally invasive detection of airways diseases especially ACO.

Antibodies are considered to be the first choice among scientists and researchers as a molecule recognition probe in biosensors due to their higher specificity and affinity towards target antigen. The antibodies are “Y” shaped molecules produced by the cell immune system, consisting of two identical polypeptide chains (Fab' fragments) and one Fc domain. Fab' fragments are mainly responsible for antigen binding where Fc fragments bind with the SAM modified electrode covalently. Covalent bonding is more suitable for antibody immobilization as it enables appropriate orientation of antibody with the binding sites and helps in facile antibody binding with SAM layer.

Development of Immunosensor:

One of the embodiment is to develop an electrochemical immunosensor on a gold working electrode for accurate diagnosis of ACO. The high sensitivity and selectivity of antigen antibody interactions have achieved incredible potential for applications in immunosensing. Immunosensing method involves the interactions between an antibody and antigen on the surface of a transducing element, that helps in measurement of electrical parameters. We propose to immobilize antibodies which can bind to specific molecules present in the serum of the patients. For the immobilization the initial step is the development of a self-assembled monolayer layer (SAM). After the formation of an orderly SAM layer, the next step would be activation of the layer followed by antibody attachment on its surface. The experiment begins with coating of SAM layer, and its characterisation using Fourier Transform Infrared (FTIR) and Atomic Force Microscopy (AFM), which has been done to generate preliminary data.

Fabrication of the immunosensor:

Gold electrodes were exposed to oxygen plasma for 10 minutes. The electrodes were then rinsed with alcohol and distilled water successively and dried in nitrogen. The pretreated electrodes were immersed in ethanol solutions of 3-mercaptopropanoic acid (3 -MPA) at four different concentrations lOmM, 20mM, 30m M and 40mM, respectively. After this, the electrodes were kept in the solution for 24 hours, at room temperature (RT) with controlled humidity conditions, to form a SAM. After ~24hrs incubation at RT, the substrates were rinsed by ethanol followed by deionized water to remove excess unbound thiols. The substrates were cleaned with nitrogen flow. They were stored in petri dishes at RT under vacuum conditions till surface characterization was done.

ATR-FTIR analysis:

The presence different functional groups at the ethanolic solution of 3MPA and SAM modified electrode has been studied using Fourier Transform Infrared (FTIR) spectroscopy in Attenuated Total Reflectance (ATR) mode. FTIR-Spectra for different concentration of 3MPA solution and SAM modified gold electrode were acquired at room temperature using Perkin Elmer Spectrum- 2 FTIR instrument.

Activation process: The SAM modified electrode were treated with 0.4 mM.EDC-O.lmM NHS for 1 hour to form EDC-NHS ester replacing the terminal carboxylic group of 3MPA SAM.

Atomic Force Microscopy (AFM):

The gold electrode surface before and after SAM layer formation was characterized using tapping mode AFM (Bruker Nanoscope V). The surface topology of the device was analysed with NanoScope Analysis for measurement of average height and average surface roughness depicted in Figures (6 and 7). This is the repletion of several experiments to get the conditions established for the better out come and reproducibility of results.

Electrochemical measurements:

All electrochemical and impedimetric measurements were taken in VersaStat4 potentiostat by Princeton Applied Research. The developed sensor was tested at each fabrication stages with 0.5 mM K3[Fe(CN)6]/K4[Fe(CN)6] as a redox probe. lOmV sinusoidal signal were used to perform impedance spectroscopy in the frequency sweep of 1 Hz to lOKHz. Measured spectra were then fitted against EC-Lab software to extract the electrical parameters out of it. It is observed that the charge transfer resistance has increased gradually after EDC-NHS ester formation and thus confirms the proper interaction of antigen- antibody at the final stage (Figure 12). EIS plot for BSA step is omitted in the Figure 12 to reduce complexity of the study.

Antibody coupling:

After formation of EDC-NHS ester the device was incubated with the target antibody for 2 hours at 37°C for antibody immobilization over the gold electrode surface. After the incubation the electrodes were rinsed with PBS solution to remove excess antibodies over the surface. The antibody immobilized electrode then incubated with 0.1% BSA to block any unspecific binding. The electrodes were finally rinsed with PBS buffer to proceed for antigen interaction.

Antigen-antibody interaction

After successful immobilization of selected antibody over gold electrode the target 50pl of target antigen were incubated for 2 hr at 37 °C. After incubation the sensor were rinsed with PBS to remove all residues. Finally, impedance spectroscopic measurements were carried out to verify the antigen antibody binding interaction.

Colorimetric analysis:

In parallel, the activated SAM layer on gold based sensor was checked to make sure IL-4 was mounted on the activated SAM layer through calorimetric assay (HRP tagged Ab). Conclusion:

The ATR-FTIR of 3-MPA in ethanol (at four different concentrations) shows formation of -C=O at 1696-1710 cm'¹ and -CH groups at 1660 cm'¹, 1042 cm'¹ and 1330 cm'¹ as expected. The alkanethiols on Au show the presence -CH band at around 1330 to 1042 cm'¹ as shown in figure (Figure 5). However, the -C=O bond at around 1696-1710 cm'¹ is not detected.

The AFM images of the gold electrode with the SAM layer has been shown in figures 6 to 7. AFM data indicates the thickness of the bar gold surface (control) to be 1.34 nm; which increases to 13.93 nm, 20.48 nm, 8.74 nm and 1.06 nm as presented in figure 6 with the formation of SAM layer corresponding to 10-, 20-, 30- and 40-mM concentrations respectively. The data also gives the roughness of bare gold surface (control), as shown in figures 7 with the formation of SAM layer at the same concentrations of 3-MPA as given in above figures.

Our research shows that gold electrode may be used for the development of IL-4 based ACO sensor. Which promises to be highly accurate and reliable sensor. The sensor shows a limit of detection (LOD) of 1 ng/ml which is superior or at par with the available reported sensor. The developed sensor may provide accurate and robust predictions which will help clinician in disease diagnosis. The sensor could also operate in the regions where limited healthcare facility is available.

In this disclosure, we have developed immunosensor for the detection of IL-4 antigen through the immobilization of Antibody on SAM modified gold electrode. The quick affinity of target antigen towards the antibody is measured in terms of electrochemical measurements. The impedance spectroscopy technique is highly sensitive and accurate as it operates in very lower voltage region and immune to any unwanted redox reaction. In future, the sensitivity of the sensor may be increased with help of different nanomaterial based immobilization of antibodies. To standardize those experiments further experimentation is needed to get reproducible results.

Preliminary SAM layer formed on gold electrodes are well characterized in our experimental set up. The mounting of antibody on activated gold electrode is confirmed through various assays. The fabrication of the device is being further standardized for reproducibility of the data with IL- 4 Antibody and experiments are underway to perform the Antigen binding assay on mounted IL- 4 Antibody on gold electrode after EDC/NHS activation. We have done a difficult part in establishing conditions for the fabrication of the immunosensor.

ADVANTAGES: The method of the present invention has various advantages. This diagnostic system and method are cost-effective, simple for use and requires less time to obtain results. The developed sensor has the potential to be the next point of care device for accurate diagnosis of ACO. The present invention provides a device for quick diagnosis with 98% average accuracy and 1 ng/ml limit of detection. Considering its good selectivity and sensitivity, the present immunosensor can be a good alternative for other conventional sensors as it is cost effective, reliable and requires low sample volume of 50pl, therefore, it can be used for rapid and simple electrochemical detection of ACO based on altered IL-4.

References:

1. Global Initiative for Asthma (GINA), global strategy for Asthma management and prevention. Available from www.ginasthma.org.2014.

2. Global Initiative for Chronic Obstructive Lung Disease (GOLD). Global strategy for diagnosis, management and prevention of COPD. Available from www.goldcopd.org. 2014

3. Miravitlles M, Soriano JB, Ancochea J, Munoz L, Duran-Tauleria E, Sanchez G, et al., Characterization of the overlap COPD-asthma phenotype. Focus on physical activity and health status. Respir Med. 2013; Jul, 107(7): 1053-60

4. Hardin M, Cho M, McDonald ML, Beaty T, Ramsdell J, Bhatt S, etal. The Clinical and genetic features of COPD-asthma overlap syndrome. Eur Respir J. 2014; Aug , 44 (2): 341-50

5. Menzes AM, Montes de Oca M, Pereez-Padilla R, Nadeau g, wehrmeister FC, Lopez- Varela MV, et al. Increased risk of exacerbation and hospitalization in subjects with an overlap phenotype: COPD-asthma. Chest. 2014; Feb,145(2):297-304.

6. Postma DS, Boezen HM. Rationale for the Dutch hypothesis. Allergy and airway hyperresponsiveness as genetic factors and their interaction with environment in the development of asthma and COPD. Chest. 2004; Aug, 126(2 Suppl): 96S- 104S;discussion 59S-61S.

7. Barnes PJ. Immunology of asthma and chronic obstructive pulmonary disease. Nat Rev Immunol. 2008; 183-192.

8. Barnes PJ. The cytokine network in asthma and chronic obstructive pulmonary disease. J Clin Invest.2008;118(l l):3546-355 9. Ghebre MA, Bafadhel M, desai D, Cohen SE, Newbold P, Rapley L, etal. Biological clustering supports both “Dutch” and "British" hypotheses of asthma and chronic obstructive pulmonary disease. J Allergy Clin Immunol. .2015 ; Jan, 135(1): 63-72

10. Newby C , Heaney LG, Menzies-GOw A, Niven RM, Mansur A, Bucknail C, et al. Statistical cluster analysis of the British Thoracic Society Severe refractory Asthma Registry: clinical outcomes and phenotype stability. PLoS One. 2014; 9(7):el0298

11. Moore WC, Meyers DA, Wenzel SE, Teague WG, Li H , Li X, et.al. Identification of asthma phenotypes using cluster analysis in the Severe Asthma Research Program. Am J Respir Crit Care Med. 2010; Feb 15, 181(4):315-23

12. Wu W, Bleecker E, Moore W, Busse WW, Castro M, Chung KF, et al. Unsupervised phenotyping of Severe Asthma Research Program participants using expanded lung data. J Allergy Clin Immunol. 2014; May, 133(5): 1280-8

13. Fu JJ, McDonald VM , Gibson PG, Simpson JL. Systemic Inflammation in Older Adults With Asthma-COPD Overlap Syndrome. Allergy Asthma Immunol Res. 2014; Jul, 6 (4);316-24

14. Iwamoto H, Gao J, Koskela J, Kinnula V, Kobayashi H, Laitinen T, et al. Differences in plasma and sputum biomarkers between COPD and COPD-asthma overlap. Eur Respir J 2014: Feb,43(2):421-9.

15. Felix FS, Angnes L. Electrochemical immunosensors -A powerful tool for analytical applications. Biosens Bioelectron. 2018; Apr 15, 102:470-478.

16. Aydin EB, Sezginturk MK. A sensitive and disposable electrochemical immunosensor for detection of SOX2, a biomarker of cancer. Talanta.2017;Sep 1, 172;162-170

17. Zeng Y, Bao J, Zhao Y, Huo D, Chen M, Yang M, Fa H,Hou C. A sensitive label- free electrochemical immunosensor for detection of cytokeratin 19 electrode. Taianta 2018; Feb 1, 178:122-128

18. Kim DM, Noh HB, Park DS, Ryu SH, Koo JS, Shim YB. Immunosensors for detection of Annexin II and MUC5AC for early diagnosis of lung cancer. BiosensBioelectron. 2009; Oct 15, 25(2): 456-462.

While the invention has been described and illustrated with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additions of procedures and protocols may be made without departing from the spirit and scope of the invention.

Claims

We Claim:

1. Process for preparing electrochemical immunosensor on a gold electrode for early and accurate diagnosis of Asthma-COPD Overlap Syndrome (ACO), said method comprising:

(f) preparing a self- assembled monolayer layer (SAM) on gold electrode by: v. exposing gold electrodes to oxygen plasma; vi. rinsing the electrode with alcohol and double distilled water successively and drying in nitrogen to obtain pre treated electrode; vii. immersing the pretreated electrode in ethanol solutions of 3- mercaptopropanoic acid (3 -MPA) at four different concentrations lOmM, 20mM, 30mM and 40mM, respectively and keeping the electrodes in the solution for 24 hours, at room temperature and controlled humidity conditions, to form a self assembled monolayer (SAM); viii. after ~24hrs incubation at room temperature, the substrates are removed and rinsed by squirting excess ethanol over them to remove unbound thiols and the substrates are blown dry with nitrogen;

(g) activation of the self-assembled monolayer layer (SAM) on gold electrode; i. treating the thiol-modified electrodes with 0.4 mM.EDC-0.lmM NHS for 15mins to 1 hour to convert the terminal carboxylic groups to an active NHS ester;

(h) antibody attachment on surface of the activated self-assembled monolayer layer (SAM) on the gold electrode; v. dropping 10-20 ng/ml of Anti-Human IL-4 onto the surface of the activated SAM at 37 °C PBS for 1-2 hr; vi. removing excess antibodies by rinsing with PBS; vii. treating the antibody-modified electrodes with 0.1-0.5% BSA for 30 mins to 1 hour, unreacted and non-specific sites; viii. after rinsing with PBS and water, drying the electrodes under nitrogen;

(i) performing impedance measurements after gold surface activation with EDC and antibody immobilization; and

(j) after Ab immobilization, non-specific binding was blocked using BSA.

2. An electrochemical immunosensor on a gold electrode for early and accurate diagnosis of Asthma-COPD Overlap Syndrome (ACO) by detecting antigen antibody complex formation in ACO associated COPD and converting it to an electrical signal for processing, recording and display; wherein ACO marker is Interleukin-4 (IL-4).

3. The immunosensor as claimed in claim 2, wherein said immunosensor differentiates ACOfrom Asthma and COPD.

4. The immunosensor as claimed in claim 2, wherein the immunosensor provides output within one minute at room temperature using 50 pl of serum sample of the subject.

5. Method for differentiating Asthma-COPD Overlap Syndrome (ACO) from Asthma and COPD, said method comprising: preparing and activating a SAM layer on gold electrodes; and mounting antibody on activated gold electrode; wherein the Interleukin-4 (IL-4) is an immune mediator and the immobilized antibody binds to the IL-4 present in the serum of the subject.