WO2020100159A1

WO2020100159A1 - A novel aptamer and an electrochemical biosensor for the rapid detection and diagnosis of tuberculous meningitis

Info

Publication number: WO2020100159A1
Application number: PCT/IN2019/050827
Authority: WO
Inventors: Tarun Kumar Sharma; Jaya Sivaswami Tyagi; Abhijeet Dhiman; Ritu Das
Original assignee: Translational Health Science And Technology Institute; All India Institute Of Medical Sciences; Aptabharat Innovation Private Limited
Priority date: 2018-11-13
Filing date: 2019-11-08
Publication date: 2020-05-22

Abstract

The invention provides a novel HspX specific aptamer, an electrochemical biosensor based on HspX specific aptamer for rapid and sensitive diagnosis of Tuberculosis meningitis and methods of detecting Tuberculous meningitis.

Description

A NOVEL APTAMER AND AN ELECTROCHEMICAL BIOSENSOR FOR THE RAPID DETECTION AND DIAGNOSIS OF TUBERCULOUS MENINGITIS

FIELD OF INVENTION

The present invention generally relates to the field of biotechnology. In particular, the present invention relates to aptamer-based electrochemical biosensor for the detection of tuberculous meningitis or tuberculosis meningitis (TBM).

BACKGROUND OF THE INVENTION

Tuberculosis (hereinafter the“TB”) is an immense public health challenge of the world with a global burden of estimated 10.4 million cases and causing 1.7 million deaths, including that of 250,000 children in the year 2016. The emergence of drug-resistant strains of TB and co- infections with human immunodeficiency virus (hereinafter the“HIV”) also add-on this burden globally. Mycobacterium tuberculosis (hereinafter the“M.tb.”) is a slow growing, obligate aerobe, non-motile, non-spore forming, and non-capsulate straight or slightly curved rod shaped bacterium. On the basis of site of infection TB is divided into two categories namely; Pulmonary (in about 90% cases it involves Lungs, and nearly 25% of people do not reflect any symptoms i.e. they remain "asymptomatic) and Extra pulmonary (when infection outburst other than lungs which involves Pleura, CNS, Lymphatic systems, bones & joints, called as extrapulmonary tuberculosis).

Tuberculous meningitis (hereinafter the“TBM”) is one of the most devastating manifestations of extra pulmonary tuberculosis having an estimated mortality of 1.5 per 100,000 population in India. In majority of TBM cases, children and immuno-compromised adults are primarily at risk of central nervous system involvement and those who remain untreated ultimately become comatose and die. More than 33% patients that receive medical care do not survive at the time of diagnosis as vast majority of TBM patients have advanced stage of disease with irreversible neurological damage. TBM involves severe neurological complications including but not limited to neurological sequelae, cerebral abscess, hydrocephalus, and other complications. Prompt diagnosis is therefore the key to improving patient survival and minimizing neurological damage. To date, TBM diagnosis remains a major challenge as the symptoms and signs at presentation are often unspecific and can resemble other meningoencephalitides, which could misguide the treatment. Besides symptoms, the conventional approaches to diagnose TBM are microscopy or culture of cerebrospinal fluid (hereinafter the“CSF”). However, these approaches have major drawbacks owing to poor sensitivity as in case of CSF smear microscopy and high turnaround time (up to eight weeks) for culture. The collection of adequate volume of CSF sample especially from pediatric patients also poses a great challenge. Although the automated PCR-based molecular test Gene Xpert MTB/RIF (Xpert) is rapid and minimal training is required for its use, its major drawback is the high cost and dependence on sophisticated instrumentation and proprietary reagents. Moreover, according to WHO 2013 meta-analysis to assess the diagnostic performance of GeneXpert for TBM, sensitivity was only 55% with 84% negative predictive value (NPV) when a clinical gold standard was used. In high prevalence TBM settings, this NPV equates to 1 in 6 TBM patients tested by Xpert being missed. Considering the current evidence therefore, the use of Xpert as the sole diagnostic test for TBM is not advisable as the treatment delay in TBM is a life-threatening condition. Alternate diagnostic approaches, including but not limited to Computed Tomography (CT) and Magnetic Resonance Imaging (MRI), do not always help in discriminating TBM from the closely related clinical conditions, especially in settings where the prevalence of TB is high. In this context, an early diagnosis could identify a large number of patients at a stage when TBM can be efficiently managed.

Importantly, the utility of detecting this protein in cerebrospinal fluid (CSF) for the diagnosis of tuberculous meningitis was established using polyclonal antibody, which yielded an excellent sensitivity and specificity of 100% and 96%, respectively. However, polyclonal antibody-based assays have inherent disadvantages of batch-to-batch variation and the use of animals for antibody generation. In-vitro synthesized DNA aptamers have enormous potential to serve as reliable tools for disease diagnosis by virtue of their specificity, high affinity, faster development time, longer shelf life and no batch-to-batch variation. Dhiman et. al. has already demonstrated in an Aptamer Linked Immobilized Sorbent Assay (ALISA) that an efficiently engineered aptamer can significantly differentiate between cerebrospinal fluid specimens from TBM and non-TBM subjects (n = 87, ***p < 0.0001) with ~ 100% sensitivity and ~91% specificity (Dhiman et al. Tuberculosis 112 (2018) 27-36). Since ALISA takes ~5 hours so to make a more rapid test in the present invention we have developed an electrochemical sensing test for TBM that takes ~30min.

Therefore, to address these formidable challenges and to be able to rule out a potentially life- threatening condition, a rapid, sensitive, low cost and simple test with reasonable specificity is urgently needed for TBM diagnosis.

OBJECT OF THE INVENTION

An object of the invention is to provide DNA aptamers, compositions and methods for detecting the presence of tuberculous meningitis or tuberculosis meningitis (TBM) through a biosensor.

SUMMARY OF THE INVENTION

The present invention provides a novel aptamer, compositions and methods for detecting the presence of tuberculous meningitis or tuberculosis meningitis (TBM) through a biosensor.

BRIEF DESCRIPTION OF FIGURES

Figure 1 provides schematic illustration of the principle of the aptamer-based electrochemical biosensor of the present invention.

Figure 2 provides Circular dichroism spectrum of HspX specific aptamer (10 mM) in absence and presence of HspX antigen (20 mM).

Figure 3 provides scanning electron micrograph of screen-printed electrode, before and after electrodeposition of gold (at 30,00K and 50,00 K magnification).

Figure 4 provides cyclic voltammogram of carbon-screen printed electrode (CSPE) before (black) and after (red) electrodeposition of GNPs.

Figure 5 provides a three-colour gradient heat-map representation of sensor response. Red colour represents highest sensor response while blue indicates the weakest sensor response.

Figure 6 provides depiction of electrochemical sensor response as a function of HspX protein and data was fitted using sigmoidal fitting equation in Origin Pro 8 software. Figure 7 provides depiction of electrochemical sensor response as a function of HspX protein spiked in pooled CSF (NIND) samples and data was fitted using sigmoidal fitting equation in Origin Pro 8 software.

Figure 8 provides Scatter plot showing sensor response for CSF samples from Definite (true positive) and NTIM (true negative) subjects (A). Receiver Operating Characteristic curve (ROC curve) derived from sensor response for Definite (true positive) and NTIM (true negative) samples (B) showing area under the curve (AUC) as 1.0 evincing robust performance of the sensor.

Figure 9 provides Scatter plot showing performance of H63 SL-2 M6 aptamer-based electrochemical sensor on archived CSF samples obtained from TBM patients (n=39; Definite n=16, Probable n=7, Possible n=16) and Not-TBM patients (n=42; NTIM n-16, IND n=16 and NIND n=10).

Figure 10 provides diagnostic performance of developed aptasensor on pleural fluid samples belonging to pleural TB and non-TB disease categories. A ROC curve was constructed using obtained values from DPV curves with pleural fluid from pleural TB, and non-TB disease.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel Aptamer. The Aptamer is represented as Seq ID 1. The Sequence of Sequence ID is:

“AGGGCTTTTTTTTTTTTTAGTTCGTTTG”

The sequence is also presented along with the Application in PATENT IN Version.

The Aptamer of the present invention is HspX specific. HspX is a potent TBM biomarker owing to its expression in-vivo and its detection by antibodies in sera collected from active TBM patients.

The Aptamer of the present invention can significantly differentiate between cerebrospinal fluid specimens from TBM and non-TBM subjects -95% sensitivity and -97.5% specificity and has the ability to deliver sample-to-answer in -30 minutes. Yet another advantage of present invention is that it is highly selective and can discriminate TB meningitis from the meningitis caused by bacterial pathogens ( E.coli and Acenitobacter spp.) other that TB. Further, it can also discriminate TBM from non -infectious neurological disorders (neurodegenerative disorders, hypocalcemic seizures and transverse myelitis, Guillain-Barre syndrome) and infectious neurological disorders (meningoencephalitis, enteric encephalopathy, sepsis, cerebral malaria, pneumonia and post diphtheritic polyneuritis).

In an embodiment, the Aptamer of the present invention may be obtained by synthetic means or using polymerase chain reaction (PCR).

In an embodiment, the present invention also discloses a biosensor containing the aptamer of Sequence ID 1. The use of the biosensor for detection of TB in the present assay is designed on a molecular switch based on efficiently engineered HspX specific aptamer for electrochemically diagnosing TBM in a human subject. The aptamer based electrochemical biosensor (hereinafter the“Aptasensor”) for the rapid in vitro diagnosis of TBM in human subject.

The Aptasensor of the present invention comprises:

(i) an aptamer modified screen printed electrode (SPE), wherein the working electrode part of SPE is coated with the Aptamer of Seq ID No.l;

(ii) a reference electrode made up of AgCl;

(iii) a potentiostat;

(iv) a tagging unit;

(v) Aptamer cartridge based on field-effect transistor (FET);

(vi) Aptamer-based acoustic sensor;

The SPE is selected from the group consisting of carbon electrode, carbon electrode coated with gold nanoparticles, platinum, preferably, the electrode is gold nanoparticle coated carbon electrode and wherein the reference electrode is AgCl.

Poteniostat is an electronic device that can be used to control a three-electrode system. It can monitor subtle change in electric current that is takes place on molecular recognition element (aptamer in this case) and its target. It can perform variety of assay using various techniques including but not limited to Differential Pulse Voltammetry (DPV), Cyclic Voltammetry (CV) and Chronoamperometry etc. The tagging unit may be any label selected from the group consisting of redox label/methylene, fluorophore, biotin, digoxigenin, gold nanoparticles, upconverting nanoparticles, enzymes etc

The Aptasensor is capable of detecting as low as lOpg of HspX in CSF samples and provides sample-to-answer in < 30 minutes with -95% sensitivity and -97.5% specificity.

In an embodiment, the present invention provides a process for preparing the biosensor. The biosensor may be obtained by the method comprising the steps of: i. by dually labeling the aptamer, preferably modifying on one end with a thiol label and other end with a redox label,

ii. reacting the dually labelled aptamer with the electrode, preferably a gold nanoparticle coated electrode;

iii. formation of a covalent bond of the aptamer with the electrode, preferably forming a gold thiol bond (Au-SH).

In another embodiment, the present invention discloses a method of in vitro detection of Tuberculosis meningitis, the method comprises the steps of: i. contacting the sample with biosensor to obtain a sample pool;

ii. incubating said sample pool with target and a binding ion to form aptamer-target complexes;

iii. separating unbound molecules present in sample matrix from the aptamer-target complexes;

iv. detecting the response, preferably by a potentiostat.

In yet an embodiment, the Aptamer of the present invention may be used for diagnosing tubleculosis meningitis. The Aptamer of the present invention may also be utilized for detection extrapulmonary TB.

In yet another embodiment, the invention provides for a kit comprising biosensor, an aptamer- based disposable cartridge along with buffer/sample diluent and a reader.

The present invention is now illustrated by means of examples. The examples are meant for illustrative purpose only and may not be construes as limiting. EXAMPLE

Example 1: Fabrication and characterization of sensing platform

For preparing of the present invention, H63 is labelled with methylene blue at 5’ end and thiol at 3’ end to give a dual labelled H63 aptamer (hereinafter the“H63 SL2-M6”). H63 SL2-M6 is immobilized on a carbon screen printed electrode modified with gold nanoparticles by incubating 10 mM of H63 SL2-M6 in binding buffer at 30°C for overnight, thereafter rinsing thoroughly with binding buffer followed by distilled water to remove the weakly adsorbed aptamer and drying. H63 SL2-M6 modified electrode was further exposed to 10 mM 2- mercaptoethanol (b-ME) for 20 minutes to block the uncovered electrode surface. Finally, the H63 SL2-M6 modified electrode was rinsed thoroughly with binding buffer and double distilled water respectively to give an aptamer modified electrode. This aptamer modified electrode consists of many molecular switches comprising H63 SL2-M6 bound with gold nanoparticle coated spherical carbon electrode through thiol moiety and a free methylene blue moiety. In presence of HspX in CSF sample, the distance between methylene blue moiety and electrode increases due to binding between H63 and HspX which leads to sharp decrease in electron transfer, hence steep drop in current. In absence of HspX in CSF sample, methylene blue moiety remains in close proximity of the electrode which causes rapid electron transfer and a good current response is observed. The steep drop in current can be monitored using EmstatBlue.

The structural change in H63 on binding with HspX has been observed by the inventors using Circular Dichroism (CD). In absence of HspX in CSF sample, a negative peak at 246 nm and two positive peaks at 220 and 272 nm were recorded which is a typical signature of stem loop/hairpin type B-DNA structure whereas in the presence of HspX, a structural change is indicated by the change in molecular ellipticity.The Aptasensor is found to be highly selective to HspX as no steep drop in current is observed in presence of M.tb. biomarkers other than HspX like MPT-51, GlcB, CFP-10, ESAT-6, Ag85C, CFP, GroES heat shock protein, LAM. As ascertained by Differential Pulse Voltammetry (DPV), the Aptasensor can detect as low as 10 pg of HspX protein in CSF sample.

Example 2: Circular Dichroism (CD) study

At first, an HspX-induced structural change in aptamer was monitored using Circular Dichroism (CD). A negative peak at 246 nm and two positive peaks at 220 and 272 nm were recorded (Figure 2), a typical signature of stem loop/hairpin type B-DNA structure. In the presence of HspX, a structural change is indicated by the change in molecular ellipticity suggesting that aptamer can work as a molecular switch for the detection of HspX

Example 3: Electrode characterization

The scanning electron-microscopic characterization of the bare SPE shows a rough surface, while electro-deposition of gold chloride leads to coating of spherical gold nanoparticles (GNPs) on the electrode surface (Figure 3). Cyclic voltammetry (CV) further confirmed the deposition of GNPs on the electrode; an increase in current was recorded on the GNP coated electrode in comparison to the bare electrode due to the increased conductivity of gold (Figure 4). These results are in concordance with the previously published reports.

Example 4: Electrochemical detection of HspX

The prepared SPE-based sensing interface was incubated with 100 ng HspX protein for 15min at room temperature. The sensing interface was incubated with 8 different proteins (HspX, ESAT-6, CFP-10, MPT-64, MPT-51, Ag85complex, GroES and CFP and LAM of Mycobacterium tuberculosis) and incubated for 15 min for determination of selectivity of the sensor. After incubation, the electrodes were washed with binding buffer carefully to remove the unbound protein. In order to detect bound protein, Differential pulse voltammetry (DPV) was performed.

Result: Thereafter, the dual labelled (MB- and thiol-labelled) H63 SL2-M6 aptamer was immobilized on the gold coated electrode and used to evaluate the specificity of the sensor. The sensor response in the presence of various Mtb antigens (HspX, ESAT-6, CFP-10, MPT- 64, MPT-51, Ag85complex, GroES and CFP and LAM) was compared and is represented as a three-colour gradient heat map (Figure 5). The sensor response is highly selective for HspX and a sharp decrease in current was observed only in presence of HspX. Such a change was not observed in presence of the other antigens that are expressed during TB infection, such as MPT- 51, GlcB, CFP-10 and ESAT-6, or secretory proteins such as Ag85C and culture filtrate proteins, or GroES heat shock protein or LAM, a major component of bacterial cell wall.

Example 5: Limit of detection

In order to determine the low-end detection limit, first a range (0.01 to 500 ng) of HspX was spiked in binding buffer and Differential Pulse Voltammetry (DPV) was performed. Further, to study the effect of sample matrix on aptamer-HspX interaction, pooled CSF samples obtained from Non-Infectious Neurological Disorder subjects (NIND) was diluted (1:10 in binding buffer) and then it was spiked with HspX (0.01 to 500 ng). Following this, DPV was performed to determine the lowest possible amount of HspX that can be detected in the CSF.

Result: The sensitivity of HspX detection using the electrochemical sensing approach was determined next. The aptamer-based electrochemical sensor was highly sensitive and able to detect as low as 10 pg of HspX (Figure 6). Following this, a range of HspX protein (0.01-500 ng) spiked into CSF (obtained from Not-TBM subjects belonging to non-inf ectious neurological disorders category, described under‘Classification of CSF samples’ in methods section) was evaluated to assess the effect of clinical sample matrix on sensor sensitivity. Notably, the aptasensor exhibited a similar low-end detection limit of 10 pg HspX in CSF background (Figure 7). However, the signal strength was marginally reduced in the CSF background, suggesting the possibility of mild quenching of the signal by the sample matrix.

Example 6: Application of the aptasensor in clinical assay

To detect HspX level in clinical samples, archived CSF samples were used as described in classification of CSF samples section. These CSF samples were diluted to 1:10 in binding buffer (10 mM Tris pH 7.5, 25 mM NaCl, 10 mM MgCh, 50 mM KC1) before performing experiment (DPV).

Result: Finally, to demonstrate the clinical diagnostic utility of the developed aptasensor, its performance was assessed in a blinded manner in 81 archived CSF specimens from pediatric subjects belonging to TBM and Not-TBM disease control categories, as described in methods section under‘Classification of CSF samples’. An ethical permission was obtained from the institutional ethics committee for the use of CSF samples in the current study. A ROC curve was constructed using DI/mA values (current difference before and after adding the sample) obtained with CSF from Definite (True positive) and NTIM group (True negative) samples. The area under the curve was 1.0 (Figure 8) and established that the assay is highly robust. The NTIM category comprised of 16 cases of pyogenic bacterial meningitis that included 14 cases that were diagnosed on the basis of response to appropriate antibiotics, clinical presentation along with symptoms and culture confirmed cases of E. coli (n=l), and Acinetobacter sp (n=l). Based on the cut-off derived from the ROC curve, the performance of electrochemical sensor was evaluated in all 81 samples (Figure 9). The test yielded a highly discriminatory response (p < 0.0001 ) for TBM and Not-TBM category with -95% sensitivity and -97.5% specificity. A recent assessment of Xpert on CSF samples showed its performance to be modest-55% sensitivity. While a newer version of Xpert MTB/RIF i.e. Xpert Ultra exhibited 70% sensitivity for probable or definite tuberculous meningitis subjects having HIV as compared to a uniform case definition. In comparison, the aptamer-based electrochemical sensor developed in this study was significantly superior with -95% sensitivity. Moreover, owing to its simple format, the developed electrochemical sensor meets the requirements of a rapid portable bedside diagnostic test for TBM. Importantly, the electrochemical sensor designed in the current study requires a very small volume of CSF (-4 pL) and addresses the challenge of collecting a large volume of CSF for various diagnostic investigations. In comparison to the existing methods, the test developed in the current study gives sample-to- answer in < 30min with high selectivity and sensitivity. Further, the developed assay can be operated through a mobile phone that makes it a truly point-of-care assay and can also help in rapid dissemination of results and clinical decision making. To evince the wider utility, we have also assessed the diagnostic performance of developed aptasensor on 63 pleural fluid samples belonging to pleural TB and non-TB disease categories. A ROC curve was constructed using obtained values from DPV curves with pleural fluid from pleural TB, and non-TB disease. The developed aptamer based electrochemical assay diagnosed pleural tuberculosis with a high sensitivity (89.47%) and moderate specificity (60%) (Figure 10). Taking together these data clearly indicate the potential utility of aptamer-based electrochemical sensor in detection various form of tuberculosis.

Claims

We Claim:

1. A HspX specific aptamer of Seq ID 1.

2. The aptamer as claimed in Claim 1, wherein the aptamer can significantly differentiate between cerebrospinal fluid specimens from TBM and non-TBM subjects -95% sensitivity and -97.5% specificity, delivers sample-to-answer in -30 minute, highly selective, discriminates TB meningitis from the meningitis caused by bacterial pathogen, discriminates TBM from non-infectious neurological disorders.

3. The aptamer as claimed in Claim 1, wherein the aptamer is obtained by synthetic means or using polymerase chain reaction (PCR).

4. A biosensor containing the aptamer of Sequence ID 1 for rapid in vitro diagnosis of TBM in human subject.

5. The biosensor as claimed in claim 4 comprising:

(i) an aptamer modified screen printed electrode (SPE), wherein the working electrode part of SPE is coated with the Aptamer of Seq ID No.1;

(ii) a reference electrode made up of AgCl;

(iii) a potentiostat;

(iv) a tagging unit;

(v) Aptamer cartridge based on field-effect transistor (FET);

(vi) Aptamer-based acoustic sensor.

6. The biosensor as claimed in claim 5, wherein the SPE is selected from the group consisting of carbon electrode, carbon electrode coated with gold nanoparticles, platinum, preferably, the electrode is gold nanoparticle coated carbon electrode and wherein the reference electrode is AgCl.

7. The biosensor as claimed in claim 5, wherein the tagging unit may be any label selected from the group consisting of redox label/methylene, fluorophore, biotin, digoxigenin, gold nanoparticles, upconverting nanoparticles, enzymes.

8. The biosensor as claimed in claim 5, wherein the biosensor can detect lOpg of HspX in CSF samples and provides sample-to-answer in < 30 minutes with -95% sensitivity and -97.5% specificity.

9. The biosensor as claimed in claim 5, wherein the biosensor of is obtained by the method comprising the steps of:

i. by dually labeling the aptamer, preferably modifying on one end with a thiol label and other end with a redox label,

10. A method of in vitro detection of Tuberculosis meningitis using the aptamer and biosensor as claimed in claims 1 to 9 comprising the steps of:

i. contacting the sample with biosensor to obtain a sample pool;

iv. detecting the response, preferably by a potentiostat.

11. Use of the aptamer and biosensor as claimed in claims 1 to 10 for detection of tuberculosis meningitis and extrapulmonary tuberculosis.

12. A kit comprising the biosensor, an aptamer-based disposable cartridge along with buffer/sample diluent and a reader.