Patent Application
20230160901
The present application relates to detection of exosomes. More particularly, the present application relates to an electrochemiluminescence nanoimmunosensor showcasing a broad linear range to detect CD63 from 100 fg mL−1 to 10 ng mL−1 in clinical samples, and a method of fabricating thereof.
One of the intracellular communications can be through nanosized vesicles, called exosomes. The exosomes are capable of transferring proteins, DNA, micro-RNA, or lipids with or without direct cell to cell. The exosomes can secrete specific proteins and nucleic acids that can cause up and/or down-regulation of physiological processes in respond to local environments, thereby facilitating tumor angiogenesis and metastasis. Such secreted proteins and nucleic acids can further be used as novel biomarkers for the detection and diagnosis of several diseases and ailments. The exosomes can be produced by most eukaryotes. Sources thereof can vary from different cells (e.g., endothelial cells, mast cell, dendritic cells, platelets, neurons, etc.) to various body fluids, such as saliva, blood, amniotic fluid, urine, breast milk, tears, and sweat in human. They also secrete a wide range of proteins including CD63, CD81, CD44, and CD69, to name a few. CD63 is the most extensively studied due to its correlation to several fatal cancers (e.g. breast cancer, ovarian cancer etc.). CD63 protein has four distinct hydrophobic domains, being associated with important cellular functions (i.e. cell development, cell activation and cell motility). In addition, the high level of CD63 protein is linked with cervical cancer, melanoma, and pancreatic cancer as well as others.
There are known conventional methods which can detect and measure the exosomal protein CD63. For example, sandwich ELISA, chemiluminescence, flow cytometry, western blotting, etc. However, the conventional methods require high throughput settings, expert technicians, and are often quite time-consuming from initial sampling until result. Furthermore, the detection of CD63 requires high sensitivity.
Therefore, there exists a need for developing immunosensors which exhibit high sensitivity and less time in spite of cheaper raw materials involved.
In a first aspect, the present application discloses a nanocomposite. The nanocomposite includes a mixture of carbon nanochips (CNCs); iron oxide (Fe3O4); and nafion (NAF). The nanocomposite includes at least 10 μg mL−1 of the CNCs; and at least 20 μg mL−1 of the Fe3O4, and at least 0.25% of the NAF in 1:1 ratio.
In a second aspect, the present application discloses an electrochemiluminescence (ECL) immunosensor. The ECL immunosensor includes an electrode modified by a nanocomposite comprising a mixture of carbon nanochips (CNCs); iron oxide (Fe3O4); and nafion (NAF). The electrode is a screen-printed electrode which further is a carbon screen-printed electrode (SPE). The carbon screen-printed electrode (SPE) is a mesoporous carbon screen-printed electrode (SPE). Ru(bpy)3Cl2.6H2O is a luminophore and tripropylamine (TPrA) is a coreactant of the luminophore. The immunosensor involves a [Ru(bpy)3]2+/TPrA complex formation between Ru(bpy)3Cl2.6H2O and TPrA. The MC-SPE/CNCs/Fe3O4/NAF electrode attracts positively charged luminophore via electrostatic interaction. The immunosensor has a CD63 detection range of 100 fg mL−1 to 10 ng mL−1.
In yet another aspect, the present application discloses a method for fabricating an electrochemiluminescence (ECL) immunosensor detecting CD63. The method involves dropping the nanocomposite over bare MC-SPF and drying thereof for at least two hours to form CNC/Fe3O4/NAF nanocomposite modified working electrode, followed by spiking anti-CD63 solution over modified-electrode/CNC/Fe3O4/NAF. The method further includes incubating the solution overnight at 4° C. to immobilize onto the modified-electrode surface by chemisorption. Thereafter, the MC-SPE/CNC/Fe3O4/NAF/anti-CD63 undergoes washing using phosphate-buffered saline (PBS) to remove loosely bound antibody and drying thereof at room temperature (RT), followed by spiking BSA over MC-SPE/CNC/Fe3O4/NAF/anti-CD63 as a blocking agent to minimize the non-specific binding and leading to formation of MC-SPE/CNC/Fe3O4/NAF/anti-CD63/BSA. The method further involves washing the MC-SPE/CNC/Fe3O4/NAF/anti-CD63/BSA using PBS and drying thereof at RT, and finally fabricating the MC-SPE/CNC/Fe3O4/NAF/anti-CD63/BSA nanoimmunosensor and storing thereof. The method involves generating light by the immunosensor as an immunocomplex forms between anti-CD63 and CD63 protein over the nanoimmunosensor.
The accompanying figures (FIGS.) illustrate embodiments and serve to explain principles of the disclosed embodiments. It is to be understood, however, that these figures are presented for purposes of illustration only, and not for defining limits of relevant applications.
For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the exemplary embodiments illustrated in the drawings, 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. Any alterations and further modifications of the inventive features illustrated herein, and any additional applications of the principles of the invention as illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the invention.
ECL is an electrochemical process between luminophore and co-reactant which produces light when a specific voltage is applied. Most commonly, the co-reactant ECL pathway is utilized for analytical detection methods. Other advantages of ECL includes the absence of background signal, flexibility thereof and suitability to many different electrode materials, sizes and dimensions, reaction position, reaction time, as well as control over applied potential and currents. There are different types of electrodes, for example screen-printed electrodes (SPE), glassy carbon electrodes (GCE), or disposable electrode printed chips (DEP-chips) which can be used in designing ECL based sensors establishing ECL as both a diverse and versatile analytical detection method. The applied voltage causes the luminophore to be oxidized, donating electrons at the electrode surface. However, as the thickness of the antibody-antigen increases over the electrode, the interactions between luminophore and electrode decrease. Further, the ECL intensity may increase and decrease depending on the net charge of the implied biomolecules on the working electrode surface.
Therefore, the present application involves utilization of ECL immunosensor for detecting CD63 by modifying existing screen-printed electrodes (SPE). In the embodiment, The ECL immunosensor includes an electrode modified by a nanocomposite. The nanocomposite includes a mixture of carbon nanochips (CNCs); iron oxide (Fe3O4); and nafion (NAF). In the embodiment, the nanocomposite includes at least 10 μg mL−1 of the CNCs; and at least 20 μg mL−1 of the Fe3O4, and at least 0.25% of the NAF in 1:1 ratio. The CNCs are two-dimensional (2D) carbon sheets resembling a similar structure of carbon nanotubes having exceptional mechanical and electrical conductivity. For instance, the CNCs reduce the rate of biofouling during analysis, and promote electron transfer on the working electrode. The Fe3O4 is well known for versatility thereof for the enhancement of sensor performance.
For the ECL immunosensor, the electrode is a screen-printed electrode which further is a carbon screen-printed electrode (SPE). The carbon screen-printed electrode (SPE) is a mesoporous carbon screen-printed electrode (SPE). Ru(bpy)3Cl2.6H2O is a luminophore and TPrA is a coreactant of the luminophore. The immunosensor involves a [Ru(bpy)3]2+/TPrA complex formation between Ru(bpy)3Cl2.6H2O and TPrA. The MC-SPE/CNCs/Fe3O4/NAF electrode attracts positively charged luminophore via electrostatic interaction. The immunosensor has a CD63 detection range of 100 fg mL−1 to 10 ng mL−1.
The NAF may be added to the CNCs/Fe3O4 composite to achieve a unique negatively charged engineered nanocomposite (CNCs/Fe3O4/NAF). The main function of the nanocomposite is to facilitate high electron transfer during the ECL reaction. Therefore, CNCs/Fe3O4/NAF nanocomposite attracts the positively charged luminophore (tris(2,2′-bipyridyl) dichlororuthenium (II) hexahydrate); Ru(bpy)3]Cl2.6H2O, over the MCSPE/CNCs/Fe3O4/NAF electrode by attractive electrostatic interaction, enabling more electron transfer between the modified-electrode surface (MC-SPE/CNCs/Fe3O4/NAF) and [Ru(bpy)3]2+/TPrA complex via redox reaction providing high ECL signal. The mechanism of the reaction between the luminophore (Ru(bpy)3]Cl2.6H2O) and the co-reactant (TPrA) and transfer of electrons is shown in
The fabricated immunosensor (MC-SPE/CNC/Fe3O4/NAF/anti-CD63/BSA) showcased notable stability and reproducibility in detecting target protein, CD63 and exhibited a wide linear range 100 fg mL−1 to 10 ng mL−1 and a low detection limit of 100 fg mL−1 for the detection of CD63.
Materials and Methods
Rabbit monoclonal antibody CD63 and exosomal protein CD63, CD81, CD69, bovine serum albumin (BSA), carcino-embryogenic antigen (CEA), alpha fetoprotein (AFP), haptoglobin (Hp), sodium azide, potassium chloride, potassium ferrocyanide, potassium ferricyanide, tris (2,2′-bipyridyl) dichlororuthenium(II) hexahydrate, tripropylamine, tris-disodium phosphate, and monosodium phosphate, nanocomposite binding agent, carbon nano chips (CNCs), 5% NAF solution were purchased from Sigma-Aldrich (USA). Iron oxide (Fe3O4) nanoparticles were procured from US Research Nanomaterials, Inc. (Houston, USA). All solutions were prepared using freshly obtained Milli-Q water (deionized with specific resistance ˜18 M cm−1). All the experiments were performed at the room temperature (RT) (21±0.5° C.).
All the ECL measurements were performed utilizing an MPI-A capillary electrophoresis electrochemiluminescence analyzer system, purchased from Xi'anYima Opto-Electrical Technology Co., Ltd. (China). A handmade ECL working cell (height 5 cm, width 1.5 cm) was utilized to detect the light generated from the reactions between ECL probe and the electrode surface. The ECL cell was placed on top of a photomultiplier tube (PMT) which was connected to the MPI-A software to analyze ECL intensity. The fabricated sensor was immersed in the ECL cell containing luminophore-coreactant mixture ([Ru(bpy)3]Cl2-TPrA) and placed on the PMT to conduct ECL measurement. The electrochemical layer-by-layer characterization studies cyclic voltammetry (CV), chronocoulometry (CC), and electrochemical impedance spectroscopy (EIS) were carried out using Autolab PGSTAT101 III potentiostat/galvanostat (Metrohm, The Netherlands) connected to a Nova software version 1.10. The disposable screen-printed electrodes were purchased from DropSens (Spain), where the working electrode was modified with mesoporous carbon, reference electrode with silver, and the counter electrode with carbon. The diameter of the mesoporous carbon modified working electrode was 4 mm. The overall dimensions of these non-reusable ceramic electrodes are (L33×H0.5×W10) mm. The surface topographical study was done by using field-emission electron microscopy (FE-SEM) JEOL, JSM-7610F (Japan). Fourier transform-infrared (FTIR) spectroscopy (Shimadzu, Japan) was used for the analysis of nanocomposite. All experiments were performed at constant room temperature (21±0.5° C.) and atmospheric pressure in an air-conditioned laboratory. All experimental data are an average of three replicates achieved from three different fabricated sensors maintaining similar optimal condition.
The preparation of the selected CNCs/Fe3O4/NAF nanocomposite was done in-house at 21±0.5° C. The CNCs and Fe3O4 were prepared in two separate small glass vials by dissolving in dH2O and were ultra-sonicated for 3.5 h for uniform dispersion. Thereafter, the sonicated nanoparticles were gradually diluted to achieve the optimum concentrations (10 mL−1 for CNCs and 20 μg mL−1 for Fe3O4). In the meantime, 0.25% NAF was also prepared by serially diluting it using double dH2O from the main stock. Finally, the synthesis of the final nanocomposite was performed by mixing 10 μg mL−1 for CNCs, 20 μg mL−1 for Fe3O4, and 0.25% NAF at 1:1 ratio and stirring it at a magnetic stirrer for 6 h. The resulting nanocomposite mixture was stored at 4° C. and was ultra-sonicated for 60 min before each use.
The present application discloses a flowchart depicting a method for fabricating the ECL immunosensor as shown in
The ECL detection of CD63 was accomplished by applying different concentrations of CD63 on MC-SPE/CNC/Fe3O4/NAF/anti-CD63/BSA nanoimmunosensor. To obtain the ECL measurements for each of the CD63 concentrations, 10 μL of CD63 was incubated on nanoimmunosenor for 60 min (at RT 21±0.5° C.) followed by washing (with 10 mM PBS, pH 7.4) and drying. The ECL detection was performed by pre-making the ECL probe mixture. The total volume of the ECL probe was 4 mL containing 1 mL of [Ru(bpy)3]2+ (1 mM) and TPrA (100 mM) each, and 2 mL of 10 mM PBS having pH 7.4. The glass cell was entirely covered with aluminum foil paper, only exposing the bottom section (diameter 1.5 cm) to authorize the diffusion of light over the PMT. The ECL cell was kept on top of the PMT which was in a lightproof black box to ensure the maximum performance of the ECL analyzer without the disturbance of external light source. All ECL measurements were performed using primary potential of 0.2 V, end potential of 1.25 V and the lowest potential as −0.2 V. The selected scan rate was 100 mVs−1 with an amplifying series of 3, sensitivity 1×10−6 and PMT potential of 800 V. Maximum ECL intensity was obtained at ˜10 s after starting each cycle. The error bars signify the relative standard deviations of at least three replicates (n=3) for all experiments.
The composition of CNCs/Fe3O4 nanocomposite was analyzed using FTIR. 2 mg of the nanocomposite sample was mixed with 200 mg KBr to form a pellet of fine consistency.
In order to confirm the structure of the nanocomposite layer on MCSPE, each nanoparticle was tested using FE-SEM. Nanoparticles were individually incubated on MC-SPE as shown in
Further, characterization of the CNCs/Fe3O4/NAF nanocomposite was performed by EC and ECL methods. The ECL produced from each layer of the nanocomposite modified on the surface of the MC-SPE was recorded to investigate the ECL enhancement observed due to the addition of the nanoparticles. The recorded ECL signal for nanoparticle and nanocomposite was compared and plotted against bare MC-SPE in
Following ECL characterization of the nanocomposite layer, the nanocomposite was subjected to electrochemical characterization, where the nanoparticle and their composite was characterized applying CV based on their response against a constant set of potential. Similar trend appeared for each layer of the nanoparticles and the composite thereof on electrochemical analysis when compared to ECL. After incubating the working electrode with CNCs, there was a significant rise in electrochemical response as shown in curve b of
Thereafter, the CC study was conducted for the MC-SPE as shown in bar a of
The optimization of antibody (anti-CD63), was accomplished by examining three different concentrations of anti-CD63 (5 μg mL−1, 1 μg mL−1 and 0.5 μg mL−1) on the MC-SPE/CNCs/Fe3O4/NAF platform at 21±0.5° C. using 100 pg mL−1 CD63 as illustrated in
The selection of the incubation time of blocking agent, BSA was achieved by preparing 0.1% BSA in 0.1% NaN3 in 10 mM PBS (pH 7.4) and 30, 60 and 90 min incubation times were verified to mitigate the nonspecific binding at the surface of MC-SPE/CNCs/Fe3O4/NAF/antiCD63/BSA immunosensor using 100 pg mL−1 CD63. The highest ECL peak was recorded at 60 min as depicted in
For the optimization of the [Ru(bpy)3]2+, different concentrations (0.5 mM, 1.0 mM and 2.0 mM), of [Ru(bpy)3]2+ were tested and 1 mM [Ru(bpy)3]2+ was found with decent stability and reproducibility. Henceforth, 1 mM [Ru(bpy)3]2+ was chosen as the optimum concentration to combine with TPrA as clearly depicted in
Both ECL and electrochemical scan rates study was performed to demonstrates the electrochemical diffusion over nanoimmunosensors.
For the layer-by-layer ECL characterization of the MC-SPE/CNCs/Fe3O4/NAF/anti-CD63/BSA nanoimmunosensor, each layer was individually incubated, and data were analyzed. Firstly, the ECL intensity for the bare MC-SPE was recorded as shown in curve a of
To evaluate the analytical performance of the immunosensor, the MC-SPE/CNCs/Fe3O4/NAF/anti-CD63/BSA platform was incubated with different concentrations (100 fg mL−1 to 10 ng mL−1) of CD63 as shown in
Further, the label-free MC-SPE/CNCs/Fe3O4/NAF/anti-CD63/BSA was authenticated for its ability to detect 100 pg mL−1 CD63, which was recorded as ECL intensity plotted against time in
The reproducibility of the developed MC-SPE/CNCs/Fe3O4/NAF/anti-CD63/BSA nanoimmunosensor was examined by fabricating five electrodes at different times and later evaluating their respective signals. The concentrations of CD63 used for testing each electrode fabrication was 100 pg mL−1 as shown in
Further elaborating analysis of SEM images shown in
As shown in
Next, Cyclic Voltammetry (CV) was performed to analyze the layers on MCSPE/CNCs/Fe3O4/NAF/anti-CD63/BSA/CD63 platform. The
Hence, the MC-SPE/CNCs/Fe3O4/NAF/antiCD63/BSA immunosensor has a wide linear range of 100 fg mL−1 to 10 ng mL−1 to detect CD63. Both CNCs and Fe3O4 provide highly accelerate ECL intensity over MC-SPE in optimal conditions. The fabricated immunosensor has good reproducibility and specificity in real-time. The immunosensor can detect other clinically important proteins including such as but not limited to CD69, and CD81.
As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means +/−5% of the stated value, more typically +/−4% of the stated value, more typically +/−3% of the stated value, more typically, +/−2% of the stated value, even more typically +/−1% of the stated value, and even more typically +/−0.5% of the stated value.
Throughout this disclosure, certain embodiments may be disclosed in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Numerous embodiments of the invention are possible. The previous exemplary embodiments are intended to merely illustrate, and not limit, the breadth and depth of embodiments that can fall within the scope of the appended claims and future claims, which define the invention. For example, the apparatus will be scaled to accommodate different flow rates of the water to be treated or impregnated. The chemical flow rates, hence the concentration of the chemistry, and the pressure in the system may be adjusted depending on the contaminants to be treated and/or the particular application.
It will be apparent that various other modifications and adaptations of the application will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the application and it is intended that all such modifications and adaptations come within the scope of the appended claims.
