Electrochemical lipidomics for cancer diagnosis

A method for detecting cancerous status of a biological sample. The method includes calculating a charge transfer resistance (Rct) of an electrochemical impedance spectroscopy (EIS) associated with lipid secretion of a biological sample, detecting a cancerous state for the biological sample responsive to the calculated Rct being equal to or more than a threshold value, and detecting a normal state for the biological sample responsive to the calculated Rct being less than the threshold value. The biological sample includes a biological sample suspected to be cancerous.

TECHNICAL FIELD

The present disclosure generally relates to cancer diagnosis, and particularly, to a nanoelectrochemical cell free approach using electrochemical impedance spectroscopy (EIS) to detect cancerous samples based on tracing the secreted lipids from a biopsied sample of a tissue.

BACKGROUND

A challenge in achieving precise diagnosis of cancer is applying external biomarkers that represent useful data from the function of disease meanwhile tumor micro-environment contains secretomes as a rich source of cancer associated macromolecules with protein, lipid and ionic natures. However, secretome analysis to trace such tumor markers requires use of complicated methods with analytical challenges and non-desired bindings in detecting the true cancer markers.

Among the secretion components, lipids are one of the crucial biomarkers. Cancer is the consequence of an alteration in lipid metabolic enzymes and pathways as far as lipid induced facilitation of metastasis become interested for cancer biologists. Cancer cells show an increased lipogenesis secreted to mediate some invasive associated pathways such as angiogenesis, immune suppressing and chemoresistance. Hence, lipids are now considered as hallmarks of cancer aggressiveness.

Hence, there is a need for a highly accurate method for cancer diagnosis via tracing lipid secretion from suspicious cell lines and biopsied samples from patients. Additionally, there is a need for a method for cancer diagnosis that should be fast, simple, and practically applicable instead of complicated methods utilizing bio-markers and long-term pathology tests. Moreover, there is a need for a cell-free method and sensor for cancer diagnosis based on lipid secretion of tissue samples instead of cell-based methods and sensors.

SUMMARY

In one general aspect, the present disclosure describes an exemplary method for detecting cancerous status of a biological sample. The method may include calculating a charge transfer resistance (Rct) of an electrochemical impedance spectroscopy (EIS) associated with lipid secretion of a biological sample, detecting a cancerous state for the biological sample responsive to the calculated Rctbeing equal to or more than a threshold value, and detecting a normal state for the biological sample responsive to the calculated Rctbeing less than the threshold value. The biological sample may include a biological sample suspected to be cancerous.

In an exemplary implementation, each of the detecting the cancerous state for the biological sample and the detecting the normal state for the biological sample may include comparing the calculated Rctwith the threshold value.

In an exemplary implementation, calculating the Rctof the EIS associated with the lipid secretion of the biological sample may include dropping peripheral aqueous media of the biological sample on an array of hydrophobic conductive nanostructures grown on three-integrated electrodes of a biosensor, recording the EIS of the dropped peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures using an electrochemical analyzer, and measuring a diameter of a semicircular curve of the recorded EIS. The peripheral aqueous media of the biological sample may include the lipid secretion of the biological sample.

In an exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of the biosensor may include dropping the peripheral aqueous media of at least one of a plurality of biological cells, a plurality of biological cell lines, a part of a tissue obtained through surgery or biopsy, a lipid phase of the biological sample, and combinations thereof on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of the biosensor.

In an exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of the biosensor may include forming a mixture of the biological sample and a solution of metal ions by mixing the biological sample with the solution of metal ions, and placing the mixture of the biological sample and the solution of metal ions on the hydrophobic conductive nanostructures array grown on the three-integrated electrodes of the biosensor.

In an exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of the biosensor may include dropping an extracted lipid phase of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of the biosensor. In an exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of the biosensor may include extracting a lipid phase from the biological sample, forming a mixture of the extracted lipid phase and a solution of metal ions by mixing the extracted lipid phase with the solution of metal ions, and placing the mixture of the extracted lipid phase and the solution of metal ions on the hydrophobic conductive nanostructures array grown on the three-integrated electrodes of the biosensor.

In an exemplary implementation, extracting the lipid phase from the biological sample may include forming a two-phase mixture of the biological sample in a solution of chloroform and methanol, separating a bottom fraction of the two-phase mixture, and drying the separated bottom fraction of the two-phase mixture. In an exemplary implementation, forming the two-phase mixture of the biological sample in the solution of chloroform and methanol may include culturing the biological sample in a cell culture media, and adding a solution of chloroform and methanol to the cultured biological sample by adding an equal volume of the solution of chloroform and methanol with a volume ratio of 1:2 (Chloroform:methanol) to the biological sample. In another exemplary implementation, forming the two-phase mixture of the biological sample in the solution of chloroform and methanol may include absorbing secretion of the biological sample by keeping the biological sample on a foam for a time period between 5 minutes and 30 minutes, forming a mixture of chloroform and the secretion of the biological sample by putting the foam containing the secretion of the biological sample in a chloroform solution inside a shaker for a time period between 5 minutes and 30 minutes, removing the foam the mixture of chloroform and the secretion of the biological sample, and adding methanol to mixture of chloroform and the secretion of the biological sample with a volume ratio of 1:2 (Chloroform:methanol).

In an exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of the biosensor may include dropping the peripheral aqueous media of the biological sample on an array of vertically aligned multi-walled carbon nanotubes (VAMWCNTs) grown on the three-integrated electrodes of the biosensor. In an exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of VAMWCNTs grown on the three-integrated electrodes of the biosensor may include dropping the peripheral aqueous media of the biological sample on the array of VAMWCNTs with a length between 2 μm and 12 μm and a diameter between 20 nm and 75 nm for each VAMWCNT of the array of VAMWCNTs.

In an exemplary implementation, recording the EIS of the dropped peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures using the electrochemical analyzer may include recording the EIS from the dropped peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures at a AC voltage between 5 mV and 10 mV by sweeping a plurality of frequency values between 0.01 Hz and 100 kHz.

In an exemplary implementation, calculating the Rctof the EIS associated with the lipid secretion of the biological sample may further include fabricating the biosensor by growing the array of hydrophobic conductive nanostructures on the three-integrated electrodes patterned on a catalyst layer deposited on a substrate. In an exemplary embodiment, the substrate may include at least one of a glass substrate, a silicon substrate, a ceramic substrate, and combinations thereof, and the catalyst layer may include a layer of at least one of iron, cobalt, nickel, and combinations thereof.

In an exemplary implementation, fabricating the biosensor may include depositing the catalyst layer on the substrate by thermally growing the catalyst layer on the substrate, patterning the three-integrated electrodes on the catalyst layer using photolithography technique, and growing the array of hydrophobic conductive nanostructures on the patterned three-integrated electrodes using a direct-current plasma enhanced chemical vapor deposition (DC-PECVD) technique. Where, the three-integrated electrodes may include a working electrode, a counter electrode, and a reference electrode.

DETAILED DESCRIPTION

In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings. The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

In an exemplary embodiment, lipids may play an important role in mediating crosstalk between cancer cells in tumor stroma. Progression in malignant phenotypes may exhibit strong correlation with increased level of lipids secreted from cancer cells. In addition, lipids may be considered as dielectric components in conductive biological media. The electrically insulating behavior of the lipids may be much stronger than other contents of intercostal and intercellular fluids.

Herein, an exemplary nanoelectrochemical cell free approach by impedance spectroscopy is disclosed that may be designed and utilized to detect cancerous samples based on tracing secreted lipids from cancer and normal samples (or cells). Accordingly, an exemplary cell-free dielectric spectroscopic method utilizing an exemplary electrochemical biosensor with three-integrated electrodes coated by a super hydrophobic electrically conductive material, such as multiwall carbon nanotube arrays (MWCNTs) may be applied to investigate the concentration of secreted lipids from the cell lines as well as biopsy samples of the patients, which may be suspicious to cancer. The exemplary MWCNTs as highly electrically conductive nanostructures with super hydrophobic properties may facilitate the perfect physical and electrical interaction between the lipids of secretion droplet and the surface of the electrodes of the exemplary electrochemical biosensor. Herein, the dielectric response of cells' secretion that may include an electrochemical impedance spectroscopy (EIS) may be compared to cells' phenotypes for any probable correlation in cancer diagnosis. The exemplary super hydrophobic electrically conductive material may form highly sensitive electrodes for EIS measurements.

FIG.1shows exemplary method100for detecting cancerous status of a biological sample, consistent with one or more exemplary embodiments of the present disclosure. Exemplary method100may include calculating a charge transfer resistance (Rct) of an electrochemical impedance spectroscopy (EIS) associated with lipid secretion of a biological sample (step102), detecting a cancerous state for the biological sample responsive to the calculated Rctbeing equal to or more than a threshold value (step104), and detecting a normal state for the biological sample responsive to the calculated Rctbeing less than the threshold value (step106). In an exemplary embodiment, the biological sample may include a biological sample suspected to be cancerous. In an exemplary embodiment, the lipid secretion of the biological sample may include a portion or whole of lipid content of the biological sample that may be extracted from the biological sample.

In detail, step102may include calculating a Rctof an EIS associated with lipid secretion of the biological sample.FIG.2shows an exemplary implementation of calculating the Rctof the EIS associated with lipid secretion of the biological sample (step102), consistent with one or more exemplary embodiments of the present disclosure. In an exemplary implementation, calculating the Rctof the EIS associated with lipid secretion of the biological sample (step102) may include dropping peripheral aqueous media of the biological sample on an array of hydrophobic conductive nanostructures grown on three-integrated electrodes of a biosensor (step204), recording the EIS of the dropped peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures using an electrochemical analyzer (step206), and measuring a diameter of a semicircular curve of the recorded EIS (step208). In an exemplary embodiment, the peripheral aqueous media of the biological sample may include secretion of the biological sample. The peripheral aqueous media of the biological sample may include the lipid secretion (lipid content) of the biological sample.

In another exemplary implementation, calculating the Rctof the EIS associated with lipid secretion of the biological sample (step102) may further include fabricating the biosensor by growing the array of hydrophobic conductive nanostructures on the three-integrated electrodes patterned on a catalyst layer deposited on a substrate (step202). In an exemplary implementation, fabricating the biosensor by growing the array of hydrophobic conductive nanostructures on the three-integrated electrodes patterned on the catalyst layer deposited on the substrate (step202) may include depositing the catalyst layer on the substrate by thermally growing the catalyst layer on the substrate, patterning the three-integrated electrodes on the catalyst layer using photolithography technique, and growing the array of hydrophobic conductive nanostructures on the patterned three-integrated electrodes using a direct-current plasma enhanced chemical vapor deposition (DC-PECVD) technique in a DC-PECVD reactor. In an exemplary embodiment, the array of hydrophobic conductive nanostructures may include an array of multi-walled carbon nanotubes (MWCNTs), for example, an array of vertically aligned multi-walled carbon nanotubes (VAMWCNTs).

FIG.3shows a schematic view of exemplary biosensor300, consistent with one or more exemplary embodiments of the present disclosure. Exemplary biosensor300may be fabricated by growing an array of hydrophobic conductive nanostructures on a three-integrated electrodes patterned on a catalyst layer deposited on a substrate (step202). Exemplary biosensor300may include exemplary substrate302and exemplary three-integrated electrodes that may include exemplary working electrode304, exemplary counter electrode306, and reference electrode308. Accordingly, the array of hydrophobic conductive nanostructures may be grown on surface of exemplary working electrode304, exemplary counter electrode306, and exemplary reference electrode308.

In an exemplary embodiment, working electrode304may have a circular shape and counter electrode306may have a ring shape around working electrode304in order to increase a resolution of the EIS that may be recorded using exemplary biosensor300that may be connected to an exemplary electrochemical analyzer.

In an exemplary embodiment, exemplary substrate302may include a substrate, on which a hydrophobic conductive material may be coated and patterned. In an exemplary embodiment, exemplary substrate302may include at least one of a glass substrate, a silicon substrate, a ceramic substrate, and combinations thereof. The silicon substrate may include a silicon substrate with a layer of silicon dioxide (SiO2) deposited thereon. In an exemplary embodiment, the catalyst layer may include a layer of a catalyst for growing the hydrophobic conductive nanostructures thereon that may include a layer of at least one of iron, cobalt, nickel, and combinations thereof. In an exemplary embodiment, the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300may include an array of VAMWCNTs with a length between about 2 μm and about 12 μm, and a diameter between about 20 nm and about 75 nm for each VAMWCNT of the array of VAMWCNTs.

Referring again toFIG.2, calculating the Rctof the EIS associated with the lipid secretion of the biological sample (step102of exemplary method100) may further include dropping peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of the biosensor that may include exemplary biosensor300(step204). In an exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300may include placing the biological sample on the array of hydrophobic conductive nanostructures by placing the biological sample on exemplary biosensor300. In an exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of the biosensor may include dropping the peripheral aqueous media of the biological sample on an array of VAMWCNTs grown on the three-integrated electrodes of the biosensor.

In an exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300(step204) may include dropping the peripheral aqueous media of at least one of a plurality of biological cells, a plurality of biological cell lines, a part of a tissue obtained through surgery or biopsy, a lipid phase of the biological sample, and combinations thereof, on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300. In an exemplary embodiment, the biological sample may include at least one of a plurality of biological cells, a portion of a tissue, a biopsied sample from a patient, a biopsied sample from a tumor, and combinations thereof. In an exemplary embodiment, the biological sample may include a biological sample suspected to be cancerous that may be examined utilizing exemplary method100for detecting presence of cancer. In an exemplary implementation, method100may be utilized for detecting the presence of at least one of breast cancer, kidney cancer, ovarian cancer, prostate cancer, skin cancer, colon cancer, stomach cancer, and combinations thereof in the biological sample.

In an exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300(step204) may include dropping whole secretion of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300. In another exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300(step204) may include dropping a lipid phase of the secretion of the biological sample, that may be derived from the biological sample, on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300.

FIG.4Ashows an exemplary implementation of dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300(step204), consistent with one or more exemplary embodiments of the present disclosure. Accordingly, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300may include forming a mixture of the biological sample and a solution of metal ions by mixing the biological sample with the solution of metal ions (step402), and placing the mixture of the biological sample and the solution of metal ions on the hydrophobic conductive nanostructures array grown on the three-integrated electrodes of exemplary biosensor300(step404). In some implementations, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300may include an individual step of placing the biological sample and the solution of metal ions on the hydrophobic conductive nanostructures array grown on the three-integrated electrodes of exemplary biosensor300.

FIG.4Bshows another exemplary implementation of dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300(step204), consistent with one or more exemplary embodiments of the present disclosure. In such exemplary implementation, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300(step204) may include dropping an extracted lipid phase of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of the biosensor. Accordingly, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300may include extracting a lipid phase from the biological sample (step410), forming a mixture of the extracted lipid phase and a solution of metal ions by mixing the extracted lipid phase with the solution of metal ions (step412), and placing the mixture of the extracted lipid phase and the solution of metal ions on the hydrophobic conductive nanostructures array grown on the three-integrated electrodes of exemplary biosensor300(step414). In some implementations, dropping the peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures grown on the three-integrated electrodes of exemplary biosensor300may include extracting the lipid phase from the biological sample (step410), and directly placing the extracted lipid phase on the hydrophobic conductive nanostructures array grown on the three-integrated electrodes of exemplary biosensor300.

In an exemplary embodiment, the solution of metal ions may include a solution containing one or more metal ions. In an exemplary embodiment, the solution of metal ions may include a solution of potassium ferricyanide (K3Fe(CN)6and/or K4Fe(CN)6) with a concentration of about 5 mM of the potassium ferricyanide in water.

In an exemplary implementation, placing the mixture of the biological sample and the solution of metal ions on the hydrophobic conductive nanostructures array grown on the three-integrated electrodes of exemplary biosensor300(step404) may include passing or flowing the mixture of the biological sample and the solution of metal ions on the hydrophobic conductive nanostructures array grown on the three-integrated electrodes of exemplary biosensor300. In a similar exemplary implementation, placing the mixture of the extracted lipid phase and the solution of metal ions on the hydrophobic conductive nanostructures array grown on the three-integrated electrodes of exemplary biosensor300(step414) may include passing or flowing the mixture of the extracted lipid phase and the solution of metal ions on the hydrophobic conductive nanostructures array grown on the three-integrated electrodes of exemplary biosensor300.

FIG.5shows an exemplary implementation of extracting the lipid phase from the biological sample (step410), consistent with one or more exemplary embodiments of the present disclosure. Accordingly, extracting the lipid phase from the biological sample (step410) may include forming a two-phase mixture of the biological sample in a solution of chloroform and methanol (step502), separating a bottom fraction of the two-phase mixture (step504), and drying the separated bottom fraction of the two-phase mixture (step506).

In an exemplary implementation, forming the two-phase mixture of the biological sample in the solution of chloroform and methanol (step502) may include culturing the biological sample in a cell culture media, and adding a solution of chloroform and methanol to the cultured biological sample by adding an equal volume of the solution of chloroform and methanol with a volume ratio of 1:2 (Chloroform:methanol) to the biological sample; thereby, equal volumes of the solution of chloroform and methanol and the biological sample may be mixed together.

In another exemplary implementation, forming the two-phase mixture of the biological sample in the solution of chloroform and methanol (step502) may include absorbing secretion of the biological sample by keeping the biological sample on a foam for a time period between 5 minutes and 30 minutes, forming a mixture of chloroform and the secretion of the biological sample by putting the foam containing the secretion of the biological sample in a chloroform solution inside a shaker for a time period between 5 minutes and 30 minutes, removing the foam the mixture of chloroform and the secretion of the biological sample, and adding methanol to mixture of chloroform and the secretion of the biological sample with a volume ratio of 1:2 (Chloroform:methanol).

As shown inFIG.2, calculating the Rctof the EIS associated with the lipid secretion of the biological sample (step102of exemplary method100) may further include recording the EIS of the dropped peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures using an electrochemical analyzer (step206). In an exemplary implementation, recording the EIS of the dropped peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures using the electrochemical analyzer (step206) may include connecting the three-integrated electrodes of exemplary biosensor300to the electrochemical analyzer, and recording the EIS from the dropped peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures. In an exemplary embodiment, the electrochemical analyzer may include a potentiostat device. In an exemplary implementation, recording the EIS from the dropped peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures may include recording the EIS from the dropped peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures at an AC voltage between about 5 mV and about 10 mV while sweeping a plurality of frequency values between about 0.01 Hz and about 100 kHz using the electrochemical analyzer.

In an exemplary implementation, recording the EIS from the dropped peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures may include recording a nyquist plot with a semicircular curve shape from the dropped peripheral aqueous media of the biological sample on the array of hydrophobic conductive nanostructures.FIG.6shows a schematic view of exemplary EIS600recorded from a dropped peripheral aqueous media of a biological sample on the array of hydrophobic conductive nanostructures, consistent with one or more exemplary embodiments of the present disclosure. Exemplary EIS600may include exemplary nyquist plot600with the semicircular curve shape including a set of recorded imaginary part of impedance (Z″(Ω)) versus a set of recorded real part of impedance (Z′(Ω)).

Referring toFIG.2, calculating Rctof the EIS associated with the lipid secretion of the biological sample (step102of exemplary method100) may further include measuring a diameter of a semicircular curve of the recorded EIS (step208). Referring toFIG.6, Rctmay be measured as a diameter of a semicircular curve of exemplary EIS600.

Referring again toFIG.1, step104may include detecting a cancerous state for the biological sample if the calculated Rctthat may be calculated utilizing step102is equal to or more than a threshold value. Moreover, step106may include detecting a normal (healthy) state for the biological sample if the calculated Rctthat may be calculated utilizing step102is less than the threshold value. In an exemplary implementation, each of detecting the cancerous state for the biological sample (step104) and detecting the normal state for the biological sample (step106) may include comparing the calculated Rctwith the threshold value.

In an exemplary implementation, the threshold value may be calculated by generating a dataset experimentally or clinically. In an exemplary implementation, generating the dataset may include generating a first set of Rctvalues that may be calculated from a plurality of normal (healthy) biological samples, generating a second set of Rctvalues that may be calculated from a plurality of cancerous biological samples, and selecting the threshold value that may include a Rctvalue at a border line between the first set of Rctvalues and the second set of Rctvalues.

In an exemplary implementation, the first set of Rctvalues and the second set of Rctvalues may be camculated utilizing exemplary process of step102for calculating the Rctof the EIS associated with lipid secretion of the biological sample described hereinabove. In an exemplary embodiment, the first set of Rctvalues may be calculated for a plurality of known normal (healthy) biological samples, for example, a plurality of healthy cell lines or patients. In an exemplary embodiment, the second set of Rctvalues may be calculated for a plurality of known cancerous biological samples, for example, a plurality of cancerous cell lines or patients.

In an exemplary implementation, generating the dataset may include generating the dataset as one of a calibration set of data, and a lookup table or plot. The dataset may be utilized to find the calculated Rctthat may be calculated in exemplary step102there in order to distinguish that the calculated Rctis equal to, more than, or less than the threshold value; thereby, resulting in detecting a cancerous state or a normal state for the biological sample (steps104and106). As a result, the cancerous status of the biological sample may be detected by comparing the calculated Rctwith the threshold value that may include one of detecting a cancerous state for a biological sample if the calculated Rctis equal to or more than the threshold value, and detecting a normal (healthy) state for the biological sample if the calculated Rctis less than the threshold value.

Example 1: Fabrication of the Biosensor

In this example, an exemplary biosensor similar to biosensor300was fabricated. The fabrication process of the device was started by coating a glass substrate with a thermally grown Ni layer, followed by patterning three electrodes similar to exemplary electrodes304,306, and308using standard photolithography. Then, the exemplary biosensor was placed in a direct-current plasma enhanced chemical vapor deposition (DC-PECVD) reactor to grow vertically aligned multi-walled carbon nanotubes (VAMWCNTs) on surface of the exemplary patterned three electrodes.

FIG.7shows a scanning electron microscopy (SEM) image of exemplary array of VAMWCNTs grown on exemplary three-integrated electrodes of the exemplary fabricated biosensor, consistent with one or more exemplary embodiments of the present disclosure. It may be observed that each CNT beam of the VAMWCNTs array may have a length range between about 2 μm and about 12 μm, and a diameter range between about 20 nm and about 75 nm. Highly ordered CNTs as seen inFIG.7may be achieved as described above with a desired pattern and geometry.

Super hydrophobic surface of CNT electrodes was analyzed by contact angle method for Dulbecco's Modified Eagle's medium (DMEM) cell culture media.FIG.8shows contact angle measurement of an exemplary surface of VAMWCNTs for exemplary droplets of lipid free DMEM (image802), DMEM containing about 10% lipid (image804), and DMEM containing about 20% lipid (image806), consistent with one or more exemplary embodiments of the present disclosure. These images may reveal the super hydrophobic surface of MWCNTs structure as the contact angle (the droplet forming angle) was about 146° for lipid free DMEM without any fetal bovine serum (FBS) (image802) meanwhile the contact angle in lipid contained (concentration of about 10%) DMEM solution was reduced to about 37° (image804). Increasing the lipid concentration to about 20% resulted in efficient spreading of the droplet with a contact angle of about 25° (image806). Such extended spreading of the lipid contained solution on the MWCNTs may present the usefulness of the CNTs (with hydrophobic surface) for lipid based dielectric measurements of the exemplary solution.

Example 2: Electrical Impedance Spectroscopy (Eis) of Cellular Secretion

Electrical impedance spectroscopy (EIS) of cellular secretion was carried out on the media of normal (MCF-10A), low grade cancerous (MCF-7), and high grade malignant (MDA-MB 231 and MDA-MB 468) breast cell lines cultured with similar concentration and vital cycles as analyzed by ANXV/PI technique. Table 1 shows results of the ANXV/PI analysis for the exemplary used cell lines whose secretion was used for lipid analysis. ANXV/PI results shows that concentration of the cells before investigating their media was about 3×105cells/well with the total volume of about 1 ml. Moreover, about 88% of the cells from each type were in a live cycle meanwhile just less than about 8% were in necrosis. It is observable from Table 1 that all of the cells showed similar vital cycles before removal of their secretions.

Cell lines (except MCF-10A) were kept in Dulbecco's modification of Eagle medium (DMEM) culture medium complimented with about 5% fetal bovine serum and about 1% penicillin/streptomycin at about 37° C. (about 5% CO2, about 95% filtered air). MCF-10A was maintained in DMEM culture medium supplemented with about 5% horse serum, about 100 μg/ml EGF, about 1 mg/ml hydrocortisone, about 1 mg/ml cholera toxin, about 10 mg/ml insulin and about 1% penicillin/streptomycin.

EIS of the secretions of the cell lines was carried out by the exemplary fabricated biosensor similar to exemplary biosensor300at about 10 mV AC voltage sweeping the frequency from about 0.01 Hz to about 100 kHz at 255 points. The EIS response was recorded using portable electrochemical analyzer in three-electrode electrochemical impedance spectroscopy (EIS) mode. In order to carry out the dielectric spectroscopy on cellular secretion, the lipid extracted from similar concentration of cell lines or the whole cell culture media for cell lines were mixed by about 0.25 ml of potassium ferricyanide (K3Fe(CN)6) and flown (placed) on exemplary VAMWCNTs covered fabricated integrated sensor. The electrodes were calibrated using K3Fe(CN)6.

FIG.9shows the EIS results of cellular secretions for various breast cell lines and DMEM media as a control sample, consistent with one or more exemplary embodiments of the present disclosure. The diameter of semicircular curves (as an indication of charge transfer resistance (Rct) related to current blocking ability of dielectric (lipid) content of the solution), was observably greater in secretomes of malignant cells (MDA-MB 231 and MDA-MB 468) compared to non-metastatic and normal cells (MCF-7 and MCF-10A).

Furthermore, the lipid free EIS responses of the secretions from all cellular phenotypes, in which the lipid content of the solutions was selectively removed by an exemplary process similar to step410of method100described hereinabove, were recorded. Both lipid contained and lipid free samples were mixed by about 0.25 ml of K3Fe(CN)6before recording the EIS responses. K3Fe(CN)6was used as an electrical background solution for electrical scanning.

FIG.10shows the EIS results of lipid free parts of the secretion obtained from various breast cell lines and DMEM media as a control sample, consistent with one or more exemplary embodiments of the present disclosure. It may be observed that EIS responses for lipid free cellular samples were mostly similar to each other, whereas EIS responses for lipid contained cellular samples as shown inFIG.9differ from each other significantly.

Table 2 shows comparative values of Rctfor whole secretion and lipid free parts of the secretion for breast cell lines and DMEM culture media as control samples. Rctin the total secretion of cancer cells is more than Rctin the total secretion normal cells meanwhile the lipid free parts of all secretions exhibit similar Rctvalues. The Rctvalues for cancerous cell lines, and especially for metastatic cell lines are much higher than Rctvalues for normal cell lines or low-grade cancerous cell lines. This reveals that the secretion of malignant breast cells contain significant amounts of electrically insulator dielectric materials (lipid component). Accordingly, dielectric responses of lipid free secretions are similar in all of cell lines.

According to the results presented inFIGS.9-10, and Table 2, it may be gleamed that just bioactive lipids of the secretions may play an important role in distinguishing responses of the EIS in the secretions of cancer cells. About one order of magnitude differences could be observed between the response of lipid free and lipid contained secretions of malignant breast cells while this difference is much lower in normal breast cells.

Moreover, EIS was recorded for the secretions of both normal and cancerous types of kidney, ovary, prostate, and skin cell lines.FIG.11shows the EIS results of normal and cancerous kidney cell lines, consistent with one or more exemplary embodiments of the present disclosure.FIG.12shows the EIS results of normal and cancerous ovary cell lines, consistent with one or more exemplary embodiments of the present disclosure.FIG.13shows the EIS results of normal and cancerous prostate cell lines, consistent with one or more exemplary embodiments of the present disclosure.FIG.14shows the EIS results of normal and cancerous skin cell lines, consistent with one or more exemplary embodiments of the present disclosure. It may be seen fromFIGS.11-14that presence of cancer cells in the cell line sample may be correlated with the indicated intensity of EIS peak. Cancerous samples may have intensified EIS peaks in comparison with normal (healthy) samples. Accordingly, a greater Rctvalue may be obtained for the cancerous samples compared with Rctvalue of normal samples.

Example 3: EIS Based Lipid Analysis of Clinical Biopsied Samples

To trace the probability of cancer involvement in clinical samples, the EIS of peripheral media fluid of samples resected by core needle biopsy (CNB) with similar sizes from more than 100 patients suspected to have breast cancer were recorded by the exemplary fabricated biosensor similar to exemplary biosensor300utilizing exemplary method100. The secreted content of similar volumes of tissues were collected by a spongy foam and the lipids of the secretion were extracted. The extracted lipids were mixed by about 0.25 ml of K3Fe(CN)6through the same protocol described for cell lines as described in EXAMPLE 2. The reference data for being a sample in cancerous or normal categories was standard hematoxylin and eosin (H&E) assay reported by a pathologist. So, a positive or negative score of each sample was due to the sample's histopathological results.

FIG.15shows EIS results of biopsied samples from patients suspected to have breast cancer, consistent with one or more exemplary embodiments of the present disclosure. A significantly distinguished border may be observed in the Rctbetween normal (healthy) and cancer samples. Lipid content in secretion of cancerous samples exhibited one order of magnitude further Rctthan normal samples (about 104versus about 103). The nearest patients in cancer and normal states were cancer involved patient ID7 and normal patient ID31 that presented strongly distinguished EIS of secretion responses. The Rctfor patient ID7 is about 14.8 kΩ and Rctfor patient ID31 is about 7.91 kΩ.

Table 3 shows the comparative values of the EIS and histopathological results of the biopsied samples that were prepared with similar sizes from the 100 patients suspected to have breast cancer. No contrast was observed between EIS and H&E results in these patients. The Rctin the secretion of normally diagnosed samples (designated by negative sign in columns EIS Secretion Sensor and Pathological Diagnosis) was less than about 8 KΩ meanwhile Rctvalue is more than 14 KΩ in cancerous samples (designated by positive sign in columns EIS Secretion Sensor and Pathological Diagnosis). So, secretion of normal samples was categorized in a short range of Rctdue to the rare concentration of lipid content in normal samples which may be in a range between 0 KΩ and 8 KΩ. Therefore, a threshold value for Rctequal to about 13 KΩ may be used as an appropriate criterion at a border line between cancerous region and normal region for analyzing biopsied samples suspected to have breast cancer in clinical tests. Accordingly, a biopsied sample with Rctvalue more than about 13 KΩ may be detected as a cancerous sample.